A turbidimetric detection kit for THP latex using a transmission and scattering method

Abstract

The invention discloses a through-scattering integrated method THP latex turbidimetric detection kit, which belongs to the field of biological detection technology. A through-scattering integrated method THP latex turbidimetric detection kit disclosed by the invention comprises a first reagent R1 and a second reagent R2; R1 comprises Hepes-NaOH buffer, NaCl, polyethylene glycol 12000, Tween-20, and a rabbit polyclonal antibody blocker; R2 comprises a buffer, Tween-20, 80nm carboxyl latex, 150nm carboxyl latex, 350nm carboxyl latex, a protective agent, and a rabbit anti-human THP polyclonal antibody. A through-scattering integrated method THP latex turbidimetric detection kit disclosed by the invention has a hardware detection speed equivalent to that of a traditional biochemical analyzer, and compared with ELISA, has a fast detection speed and low cost; it can be used in conjunction with a test line, which is more conducive to laboratory automation and promotion.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, and more particularly to a THP latex turbidimetric detection kit using a transmission and scattering integrated method. Background Art

[0002] Tamm-Horsfall protein (THP) was discovered by Tamm and Horsfall in 1951 and purified from urine. Immunofluorescence and immunoenzyme electron microscopy confirmed that it is produced in the epithelial cells of the thick ascending limb of the loop of Henle and the distal convoluted tubule of the renal tubule. It is a kidney-specific protein. Uromodulin and TH protein are the same substance and are used interchangeably in literature.

[0003] The molecular weight of TH glycoprotein is about 70KDa, and it can be glycosylated to varying degrees. Under normal circumstances, THP in renal tubular epithelial cells has two destinations: secretion from the base of the cell to the intercellular space and then into the blood; secretion from the top of the cell into the renal tubular cavity and into the urine. THP in the blood is mostly monomeric, while monomers, dimers and even polymers can be seen in urine.

[0004] THP is the most abundant protein in urine, and it has a wide range of biological functions. Studies have found that THP in the blood is related to the regulation of salt-sensitive hypertension, inflammation, and oxidative stress, and THP in urine has functions such as anti-infection, inhibition of stone formation, and immune regulation. This shows that THP in blood and urine can be used not only as a marker for kidney disease, but also as a marker for other diseases.

[0005] Clinically, THP can be used as a marker for distal tubular lesions. A decrease in THP in urine indicates lesions of various causes in the distal tubules, which can be seen in tubulointerstitial inflammation caused by upper urinary tract inflammation, infection, obstruction, autoimmune diseases, drug toxicity, copper and cadmium poisoning, etc. A transient increase in urinary THP can be seen in potassium dichromate poisoning and acute rejection after kidney transplantation; a continuous low level of urinary THP indicates that urinary stones are prone to form. Urinary THP determination helps to determine the effect of extracorporeal shock wave lithotripsy in patients with urinary tract stones and is used to study the mechanism of urinary stone formation.

[0006] When inflammation, autoimmune diseases, urinary tract obstructive diseases, etc. cause renal parenchymal damage, THP can be deposited in the renal interstitium and stimulate the body to produce corresponding autoantibodies. It is clinically used as a marker for positioning distal renal tubular lesions. The secretion of TH protein in urine varies from 50 to 150 mg per day. Literature reports that the biological reference range of TH protein in morning urine of healthy adults is 55.00-77.90 μg / mL (i.e. 55-77.9 mg / L) for males and 70.70-96.10 μg / mL (i.e. 70.7-96.1 mg / L) for females; various and various stages of renal lesions, especially various primary and secondary chronic renal lesions, can lead to urinary THP content (6.24±6.0 μg / ml) significantly lower than that of normal people, and the THP content of patients after renal transplantation (9.88±8.05 μg / ml) is also significantly lower than that of normal people. This suggests that the reduction of urinary THP content is closely related to various renal lesions. Correlation analysis of the urinary THP content of kidney disease patients with the commonly used clinical renal function indicators BUN and SCr showed that there was no significant correlation between the urinary THP concentration of 96 patients with various stages of kidney disease (compensated renal function stage, decompensated renal function stage) and their BUN and SCr; while there was a significant negative correlation between the decompensated stage (azotemia stage, uremia stage), that is, the urinary THP content decreased with the deterioration of renal function and the increase of BUN and SCr. The more severe the kidney disease, the lower the urinary THP content.

[0007] The biological reference range of TH protein in the serum of healthy adults is 102-295 ng / ml (i.e. 0.102-0.295 mg / L). Studies have shown that serum uromodulin decreases with progressive CKD (non-CKD: median 228 ng / mL; stage 1: 153 ng / mL; stage 2: 107 ng / mL; stage 3: 52 ng / mL; stage 4: 30 ng / mL; stage 5: 13 ng / mL).

[0008] The content of TH protein in urine is hundreds of times that in serum. The THP content in the blood and urine of uremia patients is 1 to 2 orders of magnitude lower than that of normal people. Therefore, a high low-end sensitivity of the reagent is required.

[0009] The commonly used detection method for THP is enzyme-linked immunosorbent assay (ELISA), which has the following main shortcomings: ① Sample preparation requires complex pretreatment before detection; this may increase experimental time and cost and reduce experimental repeatability; ② ELISA detection requires the use of a large number of standards and high-quality reagents, which makes the detection cost high; ③ The operation of ELISA detection is relatively simple, but the entire experimental process may take hours or days to complete, which may not be suitable for some scenarios that require quick results.

[0010] The all-in-one transmission and scattering detection method is a new methodology that emerged in 2020 and is an upgrade of the original latex immunoturbidimetry method. The all-in-one transmission and scattering instrument (such as Hitachi 3500, etc.) developed by Hitachi High-Tech Corporation of Japan in 2013 was launched in 2015 and obtained the NMPA medical device registration certificate in February 2019. The all-in-one transmission and scattering instrument adds a scattered light detection module based on the transmission detection of the traditional biochemical analyzer. Figure 1 That is, for the same test, the transmitted light and scattered light detection are completed within 0.7s, the signal values ​​are calculated separately, and then the results of the two are fused through software. Common knowledge in the IVD field is that scattering detection has high sensitivity, but the detection range is very narrow and cannot distinguish high values; transmission detection has low sensitivity and poor repeatability of low-value detection, but the detection range is wide and can effectively distinguish high values; the transmission and scattering integrated method uses scattering results in low-concentration areas and transmission results in high-concentration areas through result fusion. It inherits the advantages of transmission and scattering immunoturbidimetry and avoids the disadvantages, so that high-sensitivity and high-linear range detection can be obtained. The transmission and scattering integrated method can increase the sensitivity by an order of magnitude only through the hardware capabilities and software algorithms of the transmission and scattering integrated machine.

[0011] Therefore, providing a THP latex turbidimetric detection kit using a through-scattering integrated method is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0012] In view of this, the present invention provides a THP latex turbidimetric detection kit using a transmission and scattering integrated method.

[0013] In order to achieve the above object, the present invention adopts the following technical solution:

[0014] A through-scattering integrated method THP latex turbidimetric detection kit, comprising a first reagent R1 and a second reagent R2;

[0015] The first reagent R1 includes components with the following concentrations: 50-200 mM Hepes-NaOH buffer pH 6.8-7.5, NaCl 800-2400 mM, polyethylene glycol 120002 g / L, Tween-20 0.05%-2%, rabbit polyclonal antibody blocking agent 20 ml / L;

[0016] The second reagent R2 includes components with the following concentrations: 50-200mM buffer solution pH7.0-7.6; Tween-204-16ml / L; 80nm carboxyl latex 2.4-3.2g / L, 150nm carboxyl latex 1.6-2.4g / L, 350nm carboxyl latex 1.0-1.8g / L; protective agent 10-90g / L; rabbit anti-human THP polyclonal antibody 20-40mg / L.

[0017] Furthermore, the first reagent R1 and the second reagent R2 also include a preservative, and the preservative is 1 g / L sodium azide or 0.5 ml / L PC300.

[0018] Furthermore, in the second reagent R2, the buffer is Hepes-NaOH buffer, phosphate buffer or glycine-Tris buffer.

[0019] Furthermore, the protective agent is at least one of 10-30 g / L sucrose, 10-30 g / L trehalose, and 10-30 g / L fructose.

[0020] Furthermore, the THP latex turbidimetric detection kit by integrated transmission and scattering method also includes calibration products and quality control products;

[0021] The calibrator includes components at the following concentrations: 50 mM Hepes-NaOH buffer pH 7.4, THP antigen 0.02-200 mg / L, sodium chloride 200 mM, Tween-20 10 ml / L, preservative;

[0022] The quality control product includes components with the following concentrations: 50mM Hepes-NaOH buffer pH 7.4, THP antigen 0.1-60mg / L, sodium chloride 200mM, Tween-20 10ml / L, preservative;

[0023] The preservative is 0.5 ml / L PC300 or 1 g / L sodium azide.

[0024] Furthermore, the aforementioned one-piece through-scattering method THP latex turbidimetric detection kit is used in detecting THP.

[0025] Furthermore, the aforementioned through-scattering integrated method THP latex turbidimetric detection kit is used in detecting the content or level of THP in serum and / or urine.

[0026] It can be seen from the above technical solution that, compared with the prior art, the present invention discloses a THP latex turbidimetric detection kit using a through-scattering integrated method, which has the following beneficial effects:

[0027] The reagent used in the present invention is tested by the transmission-scattering integrated method on a Hitachi 3500 fully automatic biochemical analyzer, and the detection range can reach 0.01-2 mg / L (10-2000 ng / ml) for serum and 2-200 mg / L for urine; the intra-batch coefficient of variation CV≤8.0% (n=10), which can simultaneously meet the requirements for accurate quantification of TH protein in serum and urine under physiological and pathological conditions.

[0028] The present invention adopts the integrated test of transmission and scattering. Through the fusion of results, the scattering results are used in the low-concentration area, and the transmission results are used in the high-concentration area. It can simultaneously ensure the detection of high low-end sensitivity, wide linear range and wide safety zone. It can achieve the simultaneous determination of two types of samples, serum and urine, with one set of reagents. At the same time, the hardware detection speed of the integrated transmission and scattering method is equivalent to that of the traditional biochemical analyzer. Compared with ELISA, it has fast detection speed and low cost. It can be used in conjunction with the inspection line, which is more conducive to laboratory automation and promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0030] Figure 1 The attached figure is a transmission and scattering all-in-one machine;

[0031] Figure 2 The accompanying figure is a serum scattering channel calibration curve of Example 1;

[0032] Figure 3 The accompanying drawing is a serum transmission channel calibration curve of Example 1;

[0033] Figure 4 The accompanying figure is a calibration curve of the urine scattering channel of Example 1;

[0034] Figure 5 The attached figure is the urine transmission channel calibration curve of Example 1. DETAILED DESCRIPTION

[0035] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] Example 1

[0037] A through-scattering integrated method THP latex turbidimetric detection kit, comprising a first reagent R1 and a second reagent R2;

[0038] The formula of the first reagent R1 is as follows:

[0039]

[0040] The preparation method of the first reagent R1 is as follows:

[0041] Take 800ml of purified water, weigh each material in the above formula according to a volume of 1L, add them into the pure water in turn, make up to 1L after fully dissolving, filter with a 450nm filter membrane and set aside.

[0042] The formula of the second reagent R2 is:

[0043]

[0044] The second reagent R2 is prepared as follows:

[0045] (1) Latex cleaning: Use 100 mM MES-NaOH buffer, pH 5.5, centrifuge and replace the latex microspheres twice; take 10 ml of 10% mass concentration of latex microspheres 80 nm, 150 nm and 350 nm, centrifuge at 22000 rpm for 15 min, remove the supernatant, use 10 ml of the above MES-NaOH buffer to re-dissolve, then centrifuge again, remove the supernatant and re-dissolve, and complete the cleaning for use;

[0046] (2) Latex activation: Taking 10 ml of the above-mentioned washed latex as an example, weigh 50 mg of NHS (N-hydroxysuccinimide) and 30 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), dissolve them in 5 ml of the above-mentioned MES-NaOH buffer for washing, take 1 ml of each and add them to 10 ml of the three types of washed microspheres, and mix them under magnetic stirring at room temperature for 10 min;

[0047] (3) Cleaning after activation: The three latexes activated in step (2) were centrifuged at 22,000 rpm for 15 min, the supernatant was removed, and 20 ml of 50 mM Hepes-NaOH buffer (pH 7.4) was added for re-dissolution for later use;

[0048] (4) Antibody coupling: Three portions of rabbit anti-human THP polyclonal antibody were added to the three activated latex solutions reconstituted with Hepes-NaOH buffer, wherein 2 ml of antibody (3.2 g of latex VS 40 mg of antibody) was added to the 80 nm group; 2 ml of antibody (2.4 g of latex VS 40 mg of antibody) was added to the 150 nm group; and 2 ml of antibody (1.8 g of latex VS 40 mg of antibody) was added to the 350 nm group. The mixture was stirred magnetically at room temperature for 3 hours to complete the antibody coupling.

[0049] (5) Blocking and washing: After centrifuging the three latexes at 22000rpm for 15min, all the supernatants were removed. The 80nm group, 150nm group and 350nm group were respectively added with 33ml (final latex concentration of about 9.6g / L), 42ml (final latex concentration of about 7.2g / L) and 54ml (final latex concentration of about 5.4g / L) of blocking and washing solution (50mM pH7.4 Hepes-NaOH buffer, 16ml / L Tween-20, 30g / L sucrose, 10g / L trehalose, 10g / L fructose, 0.5ml / L PC300) for re-dissolution. The three latexes were mixed at a volume ratio of 1:1:1 and mixed at room temperature for 2 hours for use (final concentrations of 80nm carboxyl latex: 3.2g / L, 150nm carboxyl latex: 2.4g / L, 350nm carboxyl latex: 1.8g / L), which was the finished product R2.

[0050] Comparative Example 1

[0051] A through-scattering integrated method THP latex turbidimetric detection kit, comprising a first reagent R1 and a second reagent R2;

[0052] The formula of the first reagent R1 is as follows:

[0053]

[0054]

[0055] The preparation method of the first reagent R1 is as follows:

[0056] Take 800ml of purified water, weigh each material in the above formula according to a volume of 1L, add them into the pure water in turn, make up to 1L after fully dissolving, filter with a 450nm filter membrane and set aside.

[0057] The formula of the second reagent R2 is:

[0058]

[0059] The second reagent R2 is prepared as follows:

[0060] (1) Latex cleaning: Use 100 mM MES-NaOH buffer, pH 5.5, centrifuge and replace the latex microspheres twice; take 10 ml of 10% mass concentration of latex microspheres 80 nm, 150 nm and 350 nm, centrifuge at 22000 rpm for 15 min, remove the supernatant, use 10 ml of the above MES-NaOH buffer to re-dissolve, then centrifuge again, remove the supernatant and re-dissolve, and complete the cleaning for use;

[0061] (2) Latex activation: Taking 10 ml of the above-mentioned washed latex as an example, weigh 50 mg of NHS (N-hydroxysuccinimide) and 30 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), dissolve them in 5 ml of the above-mentioned MES-NaOH buffer for washing, take 1 ml of each and add them to 10 ml of the three types of washed microspheres, and mix them under magnetic stirring at room temperature for 10 min;

[0062] (3) Cleaning after activation: The three latexes activated in step (2) were centrifuged at 22,000 rpm for 15 min, the supernatant was removed, and 20 ml of 50 mM Hepes-NaOH buffer (pH 7.4) was added for re-dissolution for later use;

[0063] (4) Antibody coupling: Three portions of rabbit anti-human THP polyclonal antibody were added to the three activated latex solutions reconstituted with Hepes-NaOH buffer, wherein 0.5 ml antibody (3.2 g latex VS 10 mg antibody) was added to the 80 nm group; 0.5 ml antibody (2.4 g latex VS 10 mg antibody) was added to the 150 nm group; and 0.5 ml antibody (1.8 g latex VS 10 mg antibody) was added to the 350 nm group. The mixture was magnetically stirred at room temperature for 3 hours to complete the antibody coupling.

[0064] (5) Blocking and washing: After centrifuging the three latexes at 22000rpm for 15min, all the supernatants were removed. The 80nm group, 150nm group and 350nm group were respectively added with 33ml (final latex concentration of about 9.6g / L), 42ml (final latex concentration of about 7.2g / L) and 54ml (final latex concentration of about 5.4g / L) of blocking and washing solution (50mM pH7.4 Hepes-NaOH buffer, 16ml / L Tween-20, 30g / L sucrose, 10g / L trehalose, 10g / L fructose, 0.5ml / L PC300) for re-dissolution. The three latexes were mixed at a volume ratio of 1:1:1 and mixed at room temperature for 2 hours for use (final concentrations of 80nm carboxyl latex: 3.2g / L, 150nm carboxyl latex: 2.4g / L, 350nm carboxyl latex: 1.8g / L), which was the finished product R2.

[0065] Comparative Example 2

[0066] A through-scattering integrated method THP latex turbidimetric detection kit, comprising a first reagent R1 and a second reagent R2;

[0067] The formula of the first reagent R1 is as follows:

[0068]

[0069] The preparation method of the first reagent R1 is as follows:

[0070] Take 800ml of purified water, weigh each material in the above formula according to a volume of 1L, add them into the pure water in turn, make up to 1L after fully dissolving, filter with a 450nm filter membrane and set aside.

[0071] The formula of the second reagent R2 is:

[0072]

[0073]

[0074] The second reagent R2 is prepared as follows:

[0075] (1) Latex cleaning: Use 100 mM MES-NaOH buffer, pH 5.5, centrifuge and replace the latex microspheres twice; take 10 ml of 10% mass concentration of latex microspheres 80 nm, 150 nm and 350 nm, centrifuge at 22000 rpm for 15 min, remove the supernatant, use 10 ml of the above MES-NaOH buffer to re-dissolve, then centrifuge again, remove the supernatant and re-dissolve, and complete the cleaning for use;

[0076] (2) Latex activation: Taking 10 ml of the above-mentioned washed latex as an example, weigh 50 mg of NHS (N-hydroxysuccinimide) and 30 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), dissolve them in 5 ml of the above-mentioned MES-NaOH buffer for washing, take 1 ml of each and add them to 10 ml of the three types of washed microspheres, and mix them under magnetic stirring at room temperature for 10 min;

[0077] (3) Cleaning after activation: The three latexes activated in step (2) were centrifuged at 22,000 rpm for 15 min, the supernatant was removed, and 20 ml of 50 mM Hepes-NaOH buffer (pH 7.4) was added for re-dissolution for later use;

[0078] (4) Antibody coupling: Three portions of rabbit anti-human THP polyclonal antibody were added to the three activated latex solutions reconstituted with Hepes-NaOH buffer, wherein 2.45 ml of antibody (3.2 g of latex VS 50 mg of antibody) was added to the 80 nm group; 2.5 ml of antibody (2.4 g of latex VS 50 mg of antibody) was added to the 150 nm group; and 2.5 ml of antibody (1.8 g of latex VS 50 mg of antibody) was added to the 350 nm group. The mixture was magnetically stirred at room temperature for 3 hours to complete the antibody coupling.

[0079] (5) Blocking and washing: After centrifuging the three latexes at 22000rpm for 15min, all supernatants were removed. 33ml (final latex concentration of about 9.6g / L), 42ml (final latex concentration of about 7.2g / L), and 54ml (final latex concentration of about 5.4g / L) of blocking and washing solution (50mM pH7.4 Hepes-NaOH buffer, 16ml / L Tween-20, 30g / L sucrose, 10g / L trehalose, 10g / L fructose, 0.5ml / L PC300) were added to the 80nm group, 150nm group, and 350nm group, respectively, for re-dissolution. The three latexes were mixed at a volume ratio of 1:1:1 and mixed at room temperature for 2 hours for use (final concentrations of 80nm carboxyl latex: 3.2g / L, 150nm carboxyl latex: 2.4g / L, and 350nm carboxyl latex: 1.8g / L), which was the finished product R2.

[0080] Example 2

[0081] A through-scattering integrated method THP latex turbidimetric detection kit, comprising a first reagent R1 and a second reagent R2;

[0082] The formula of the first reagent R1 is as follows:

[0083]

[0084]

[0085] The preparation method of the first reagent R1 is as follows:

[0086] Take 800ml of purified water, weigh each material in the above formula according to a volume of 1L, add them into the pure water in turn, make up to 1L after fully dissolving, filter with a 450nm filter membrane and set aside.

[0087] The formula of the second reagent R2 is:

[0088]

[0089] The second reagent R2 is prepared as follows:

[0090] (1) Latex cleaning: Use 100 mM MES-NaOH buffer, pH 5.5, centrifuge and replace the latex microspheres twice; take 10 ml of 10% mass concentration of latex microspheres 80 nm, 150 nm and 350 nm, centrifuge at 22000 rpm for 15 min, remove the supernatant, use 10 ml of the above MES-NaOH buffer to re-dissolve, then centrifuge again, remove the supernatant and re-dissolve, and complete the cleaning for use;

[0091] (2) Latex activation: Taking 10 ml of the above-mentioned washed latex as an example, weigh 50 mg of NHS (N-hydroxysuccinimide) and 30 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), dissolve them in 5 ml of the above-mentioned MES-NaOH buffer for washing, take 1 ml of each and add them to 10 ml of the three types of washed microspheres, and mix them under magnetic stirring at room temperature for 10 min;

[0092] (3) Cleaning after activation: The three latexes activated in step (2) were centrifuged at 22,000 rpm for 15 min, the supernatant was removed, and 20 ml of 100 mM glycine-Tris buffer, pH 7.0, was added for re-dissolution and used for later use;

[0093] (4) Antibody coupling: Take 3 portions of rabbit anti-human THP polyclonal antibody and add them to the three activated latex solutions reconstituted with glycine-Tris buffer mentioned above, wherein 1 ml of antibody (2.8 g latex VS 20 mg antibody) is added to the 80 nm group; 1 ml of antibody (2.0 g latex VS 20 mg antibody) is added to the 150 nm group; and 1 ml of antibody (1.6 g latex VS 20 mg antibody) is added to the 350 nm group. Magnetic stirring was performed at room temperature for 3 hours to complete the antibody coupling.

[0094] (5) Blocking and washing: After centrifuging the three latexes at 22000 rpm for 15 min, all supernatants were removed. 37.7 ml (final latex concentration of about 8.4 g / L), 50.4 ml (final latex concentration of about 6.0 g / L), and 60.75 ml (final latex concentration of about 4.8 g / L) of blocking and washing solution (100 mM pH 7.0 glycine-Tris buffer, 10 ml / L Tween-20, 10 g / L sucrose, 30 g / L trehalose, 20 g / L fructose, 0.5 ml / L PC300) were added to the 80 nm group, 150 nm group, and 350 nm group, respectively, for re-dissolution. The three latexes were mixed at a volume ratio of 1:1:1 and mixed at room temperature for 2 hours for use (final concentrations of 80 nm carboxyl latex 2.8 g / L, 150 nm carboxyl latex 2.0 g / L, and 350 nm carboxyl latex 1.6 g / L), which was the finished product R2.

[0095] Example 3

[0096] A through-scattering integrated method THP latex turbidimetric detection kit, comprising a first reagent R1 and a second reagent R2;

[0097] The formula of the first reagent R1 is as follows:

[0098]

[0099] The preparation method of the first reagent R1 is as follows:

[0100] Take 800ml of purified water, weigh each material in the above formula according to a volume of 1L, add them into the pure water in turn, make up to 1L after fully dissolving, filter with a 450nm filter membrane and set aside.

[0101] The formula of the second reagent R2 is:

[0102]

[0103]

[0104] The second reagent R2 is prepared as follows:

[0105] (1) Latex cleaning: Use 100 mM MES-NaOH buffer, pH 5.5, centrifuge and replace the latex microspheres twice; take 10 ml of 10% mass concentration of latex microspheres 80 nm, 150 nm and 350 nm, centrifuge at 22000 rpm for 15 min, remove the supernatant, use 10 ml of the above MES-NaOH buffer to re-dissolve, then centrifuge again, remove the supernatant and re-dissolve, and complete the cleaning for use;

[0106] (2) Latex activation: Taking 10 ml of the above-mentioned washed latex as an example, weigh 50 mg of NHS (N-hydroxysuccinimide) and 30 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), dissolve them in 5 ml of the above-mentioned MES-NaOH buffer for washing, take 1 ml of each and add them to 10 ml of the three types of washed microspheres, and mix them under magnetic stirring at room temperature for 10 min;

[0107] (3) Cleaning after activation: The three latexes activated in step (2) were centrifuged at 22,000 rpm for 15 min, the supernatant was removed, and 20 ml of 200 mM pH 7.6 phosphate buffer was added for re-dissolution for later use;

[0108] (4) Antibody coupling: Three portions of rabbit anti-human THP polyclonal antibody were added to the three activated latex solutions reconstituted with phosphate buffer, wherein 1.5 ml of antibody (2.4 g latex VS 30 mg antibody) was added to the 80 nm group; 1.5 ml of antibody (1.6 g latex VS 30 mg antibody) was added to the 150 nm group; and 1.5 ml of antibody (1.0 g latex VS 30 mg antibody) was added to the 350 nm group. The mixture was magnetically stirred at room temperature for 3 hours to complete the antibody coupling.

[0109] (5) Blocking and washing: After centrifuging the three latexes at 22,000 rpm for 15 min, all supernatants were removed. 44 ml (final latex concentration of about 7.2 g / L), 63 ml (final latex concentration of about 4.8 g / L), and 97.2 ml (final latex concentration of about 3.0 g / L) of blocking and washing solution (200 mM pH 7.6 phosphate buffer, 8 ml / L Tween-20, 20 g / L sucrose, 20 g / L trehalose, 30 g / L fructose, 1 g / L sodium azide) were added to the 80 nm group, 150 nm group, and 350 nm group, respectively, for re-dissolution. The three latexes were mixed at a volume ratio of 1:1:1 and then mixed at room temperature for 2 hours for use (final concentrations of 2.4 g / L for 80 nm carboxyl latex, 1.6 g / L for 150 nm carboxyl latex, and 1.0 g / L for 350 nm carboxyl latex), which was the finished product R2.

[0110] Example 4 Calibration Curve Acquisition

[0111] Preparation of diluent (1L): Take 800ml purified water, weigh 11.9155g Hepes, 11.688g NaCl, 10ml Tween-20, and 0.5ml proclin300 respectively, put them into purified water, adjust the pH to 7.4±0.1 with NaOH after fully dissolving, make the volume to 1L, and filter with 0.22μm filter membrane to obtain the calibration and quality control dilution solution.

[0112] THP antigen (purchased from Shanghai Yunmai Biotechnology Co., Ltd.) was used to prepare calibration and quality control products according to the following concentrations:

[0113] Calibrators: 0.02mg / L, 0.08mg / L, 0.3mg / L, 0.6mg / L, 1.2mg / L, 2.0mg / L; 8.0mg / L, 20mg / L, 50mg / L, 100mg / L, 200mg / L.

[0114] Quality control products: 0.1mg / L, 1.0mg / L, 10mg / L, 60mg / L.

[0115] The reagents of Example 1, Comparative Example 1, Comparative Example 2, Example 2 and Example 3 (hereinafter referred to as reagent A, reagent B, reagent C, reagent D and reagent E) and the calibration quality control were calibrated on the machine to obtain the calibration curve.

[0116] Among them, the serum channel calibration curve of Example 1 is shown in Figure 2 (scattering) and Figure 3 (Transmission); urine channel calibration curve see Figure 4 (scattering) and Figure 5 (transmission).

[0117] The instrument used is Hitachi's all-in-one penetrating and scattering machine, taking Hitachi3500 as an example, and the method is the all-in-one penetrating and scattering method;

[0118] Machine parameters (serum):

[0119] Sample volume 20μl, R1 added volume 165μl; R2 added volume 55μl;

[0120] Transmission wavelength 600nm; scattering angle 20 degrees; two-point endpoint method; 18-34 reading points;

[0121] Scattering calibration points: 0.000, 0.02, 0.08, 0.3, 0.6;

[0122] Transmission calibration points: 0.000, 0.08, 0.3, 0.6, 1.2, 2;

[0123] Through-scatter fusion zone 0.08-0.6 mg / L;

[0124] Results less than 0.08 mg / L are reported as scattering results, results greater than or equal to 0.6 mg / L are reported as transmission results, and results within the fusion zone are reported as scattering results first.

[0125] Machine parameters (urine):

[0126] Sample volume 2μl, R1 added volume 165μl; R2 added volume 55μl;

[0127] Transmission wavelength 600nm; scattering angle 20 degrees; two-point endpoint method; 18-34 reading points;

[0128] Scattering calibration points: 0.00, 2, 8, 20, 50;

[0129] Transmission calibration points: 0.00, 8, 20, 50, 100, 200;

[0130] Through-scatter fusion zone 8-50mg / L;

[0131] Results less than 8 mg / L are reported as scattering results, results greater than or equal to 50 mg / L are reported as transmission results, and results within the fusion zone are reported as scattering results first.

[0132] The calibration data are shown in Tables 1 to 5.

[0133] Table 1-1

[0134]

[0135] Table 1-2

[0136]

[0137] Table 2-1

[0138]

[0139] Table 2-2

[0140]

[0141] Table 3-1

[0142]

[0143] Table 3-2

[0144]

[0145]

[0146] Table 4-1

[0147]

[0148] Table 4-2

[0149]

[0150] Table 5-1

[0151]

[0152] Table 5-2

[0153]

[0154] Example 5 Linear range detection

[0155] Take a calibrator (H) close to the upper limit of the linear range of urine (200 mg / L) and a calibrator (L) close to the lower limit of the linear range of urine (2 mg / L) to form 6 test specimens covering the linear range of the kit, and use reagents A, B, C, D and E to measure 3 times each according to the standard experimental operation steps. The serum linear dilution operation is the same as above, only the sample is changed to 2 mg / L and 0.02 mg / L. The test results are shown in Tables 6-10.

[0156] Table 6-1

[0157]

[0158] Note ①: Results less than 8 mg / L are reported as scattering results, and results greater than or equal to 50 mg / L are reported as transmission results. Results within the fusion zone are reported as scattering results first.

[0159] Note ②: In the sample mixing ratio, taking H as 500μl as an example, 4H+L is a mixture of 400μl H+100μl L. Similarly, 3H+2L is a mixture of 300μl H+200μl L. The same applies to other concentrations. Subsequent linear dilution operations are the same as this step.

[0160] Table 6-2

[0161]

[0162] Note: Results less than 0.08 mg / L are reported as scattering results, and results greater than or equal to 0.6 mg / L are reported as transmission results. Results within the fusion zone are reported as scattering results first.

[0163] Table 7-1

[0164]

[0165] Note: Results less than 8 mg / L are reported as scattering results, and results greater than or equal to 50 mg / L are reported as transmission results. Results within the fusion zone are reported as scattering results first.

[0166] Table 7-2

[0167]

[0168]

[0169] Note: Results less than 0.08 mg / L are reported as scattering results, and results greater than or equal to 0.6 mg / L are reported as transmission results. Results within the fusion zone are reported as scattering results first.

[0170] Table 8-1

[0171]

[0172] Note: Results less than 8 mg / L are reported as scattering results, and results greater than or equal to 50 mg / L are reported as transmission results. Results within the fusion zone are reported as scattering results first.

[0173] Table 8-2

[0174]

[0175] Note: Results less than 0.08 mg / L are reported as scattering results, and results greater than or equal to 0.6 mg / L are reported as transmission results. Results within the fusion zone are reported as scattering results first.

[0176] Table 9-1

[0177]

[0178] Note: Results less than 8 mg / L are reported as scattering results, and results greater than or equal to 50 mg / L are reported as transmission results. Results within the fusion zone are reported as scattering results first.

[0179] Table 9-2

[0180]

[0181] Note: Results less than 0.08 mg / L are reported as scattering results, and results greater than or equal to 0.6 mg / L are reported as transmission results. Results within the fusion zone are reported as scattering results first.

[0182] Table 10-1

[0183]

[0184] Note: Results less than 8 mg / L are reported as scattering results, and results greater than or equal to 50 mg / L are reported as transmission results. Results within the fusion zone are reported as scattering results first.

[0185] Table 10-2

[0186]

[0187] Note: Results less than 0.08 mg / L are reported as scattering results, and results greater than or equal to 0.6 mg / L are reported as transmission results. Results within the fusion zone are reported as scattering results first.

[0188] It is not difficult to see from the above linear dilution results that Examples 1, 2 and 3 meet the linear dilution measured value and theoretical value deviation less than ±6% in the range of serum 0.01-2 mg / L (10-2000 ng / ml) and urine 2-200 mg / L, and the linear relationship is very good. Comparative Examples 1 and 2 both have the phenomenon of non-linear dilution, where Comparative Example 1 shows a low high value test, and Comparative Example 2 shows that the lower the dilution, the lower the measured value, and the deviation is very large.

[0189] Example 6 Repeatability Study

[0190] The first reagent R1 and the second reagent R2 prepared in Examples 1, 2, 3 and Comparative Examples 1 and 2 were used to repeat the measurement of 0.100 mg / L samples 10 times, and the average value (M) and standard deviation (SD) of the 10 measurement results were calculated. The coefficient of variation was obtained according to the following formula: CV = SD / M × 100%. In the formula: CV is the coefficient of variation; SD is the standard deviation of the 10 measurement results; M is the average value of the 10 measurement results. (Note: Because the concentration of THP in serum is a difficult-to-measure protein at the ng level, and the concentration in urine is a higher concentration protein at the mg level, the repeatability study here only tests serum low-value samples.)

[0191] The results are shown in Tables 11 and 12.

[0192] Table 11

[0193]

[0194] Table 12

[0195]

[0196]

[0197] From the above data, it can be seen that the repeatability results of Example 1, Example 2 and Example 3 at 0.1 mg / L are better than those of Comparative Example 1 and Comparative Example 2; Example 1 has the best repeatability result at 0.1 mg / L; However, as mentioned above, the transmission channel of Example 1 has insufficient sensitivity, and the CV is greater than 5%, while the CV of the scattering channel is within 2%, which further illustrates the advantages and improvements of the transmission-scattering integrated method compared with traditional transmission detection. Combined with the linear dilution results, the subsequent stability experiment was carried out on the basis of the reagents of Example 1.

[0198] Example 7 Stability Observation

[0199] 1) Accelerated stability: The first reagent R1 and the second reagent R2 prepared in Example 1 were placed in a 37°C thermostat and a 4°C refrigerator, respectively (R1 and R2 were placed in a 37°C thermostat as a group; R1 and R2 were placed in a 4°C refrigerator as a group), and the 37°C reagent and the refrigerated reagent were taken out on the 3rd, 5th and 8th days for simultaneous testing. The high / low value quality control products (concentrations of 0.1 mg / L and 1.0 mg / L, respectively) were tested separately, and the test was repeated 3 times, and the average value was taken. The average value of the accelerated reagent test result was compared with the refrigerated reagent test result, and the relative deviation (%) was calculated.

[0200] Note: 1. After the thermal acceleration reagent is taken out of the incubator, the temperature must be balanced with the refrigerated reagent to the same temperature before testing (to avoid incorrect experimental results caused by different temperatures). 2. Because THP concentration in serum is a difficult-to-measure protein at the ng level, while in urine it is a higher concentration protein at the mg level, the stability study here only uses serum parameters for testing.

[0201] The results are shown in Table 13.

[0202] Table 13

[0203]

[0204]

[0205] 2) Opening stability: After opening the first reagent R1 and the second reagent R2 prepared in Example 1, place them in a fully automatic biochemical analyzer (2-8°C), test the high / low quality control products (concentrations are 0.1 mg / L and 1.0 mg / L, respectively), and take out the open bottle reagents and the refrigerated reagents for testing at the same time on the 3rd, 5th, 7th, and 15th days. Repeat the test 3 times, take the average value, compare the average value of the open bottle reagent test results with the refrigerated reagent test results, and calculate the relative deviation (%). The results are shown in Table 14.

[0206] Table 14

[0207]

[0208] It can be seen from the results in Tables 13 and 14 that the reagent of the present invention can be stable for at least 7 days at a high temperature of 37 degrees and can be stable for at least 14 days at 4 degrees under unsealed conditions, indicating that the stability is very good.

[0209] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one 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 present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A through-scattering integrated method THP latex turbidimetric detection kit, characterized in that: It includes a first reagent R1 and a second reagent R2; The first reagent R1 includes components with the following concentrations: 50-200 mM Hepes-NaOH buffer pH 6.8-7.5, NaCl 800-2400 mM, polyethylene glycol 12000 2 g / L, Tween-20 0.05%-2%, rabbit polyclonal antibody blocking agent 20 ml / L; The second reagent R2 includes components with the following concentrations: 50-200mM buffer solution pH 7.0-7.6; Tween-20 4-16ml / L; 80nm carboxyl latex 2.4-3.2g / L, 150nm carboxyl latex 1.6-2.4g / L, 350nm carboxyl latex 1.0-1.8g / L; protective agent 10-90g / L; rabbit anti-human THP polyclonal antibody 20-40mg / L.

2. A through-scattering integrated THP latex turbidimetric detection kit according to claim 1, characterized in that: The first reagent R1 and the second reagent R2 also include a preservative, and the preservative is 1 g / L sodium azide or 0.5 ml / L PC300.

3. A through-scattering integrated method THP latex turbidimetric detection kit according to claim 1 or 2, characterized in that: In the second reagent R2, the buffer is Hepes-NaOH buffer, phosphate buffer or glycine-Tris buffer.

4. A through-scattering integrated method THP latex turbidimetric detection kit according to claim 3, characterized in that: The protective agent is at least one of 10-30 g / L sucrose, 10-30 g / L trehalose, and 10-30 g / L fructose.

5. A through-scattering integrated method THP latex turbidimetric detection kit according to claim 4, characterized in that: Also includes calibrators and quality controls; The calibrator includes components at the following concentrations: 50 mM Hepes-NaOH buffer pH 7.4, THP antigen 0.02-200 mg / L, sodium chloride 200 mM, Tween-20 10 ml / L, preservative; The quality control product includes components with the following concentrations: 50mM Hepes-NaOH buffer pH 7.4, THP antigen 0.1-60mg / L, sodium chloride 200mM, Tween-20 10ml / L, preservative; The preservative is 0.5 ml / L PC300 or 1 g / L sodium azide.

6. Use of the through-scattering integrated THP latex turbidimetric detection kit according to claim 1 in detecting THP.

7. Use of the through-scattering integrated method THP latex turbidimetric detection kit according to claim 1 in detecting THP in serum and / or urine.

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