A progesterone detection kit, preparation and application method

Abstract

The present invention provides a progesterone detection kit, preparation and application method, including three-component solid reagents; the three-component solid reagents include R1 reagent, R2 reagent and R3 reagent; the R1 reagent is a progesterone antibody coated with latex microspheres containing fluorescent and dye substances; the R2 reagent is a progesterone peptide modified with a water-soluble biotin derivative; the R3 reagent is streptavidin coated with latex microspheres containing photosensitive substances; the preparation method of the three-component solid reagent is: the R1 reagent, the R2 reagent and the R3 reagent are diluted with a diluent according to a certain concentration and then freeze-dried. The kit does not require a sequential reaction sequence and a cleaning step, nor does it require a dissociating agent, etc., and is simple to operate.

Description

Technical Field

[0001] The invention relates to a progesterone detection kit, a preparation method and an application method thereof, and belongs to the technical field of biomedical detection. Background Art

[0002] Progesterone is an important progestogen, a steroid hormone, mainly produced by the corpus luteum of the ovary and the placenta during pregnancy. It is a precursor of testosterone, estrogen and adrenal cortex hormone. The main function of progesterone is to prepare the uterus for the implantation of the fertilized egg and maintain pregnancy. In the follicular phase, the progesterone level is very low, while the amount of progesterone produced by the corpus luteum increases after ovulation and reaches the maximum concentration 5-7 days after ovulation. If there is no conception, the corpus luteum will gradually shrink and the progesterone value will decrease 4 days before the end of the menstrual cycle; if conception occurs, the corpus luteum will not shrink, but continue to secrete progesterone, and the progesterone level will remain at a certain value and continue until the 6th week of pregnancy. In clinical practice, the detection of progesterone is mainly used to assist in the screening of diseases such as threatened abortion, corpus luteum insufficiency, placental dysplasia, and hydatidiform mole.

[0003] The main methods for detecting progesterone are enzyme-linked immunosorbent assay and chemiluminescent immunoassay. Chemiluminescent immunoassay is the most commonly used progesterone detection method in clinical practice, with the characteristics of high sensitivity, strong specificity, simplicity and rapidity. Currently, most of the progesterone quantitative detection kits on the market are magnetic particle chemiluminescence methods, and the detection principle is the competition method. The competition method generally requires that the antigen to be tested reacts before the antibody, and the carrier antigen then competes, or the antigen to be tested and the carrier antigen are first mixed and then the antibody is added for simultaneous competition. The competition method has a sequential reaction order and a washing step. Some manufacturers' reagents also require dissociation agents, etc. The operation is relatively complicated, which brings inconvenience to progesterone detection. Summary of the invention

[0004] The present invention provides a progesterone detection kit, and a preparation and application method thereof, which can effectively solve the above-mentioned problems.

[0005] The present invention is achieved in that:

[0006] A progesterone detection kit comprises three-component solid reagents; the three-component solid reagents comprise R1 reagent, R2 reagent and R3 reagent; the R1 reagent is a progesterone antibody coated with latex microspheres containing fluorescent and dye substances; the R2 reagent is a progesterone peptide modified with a water-soluble biotin derivative; the R3 reagent is streptavidin coated with latex microspheres containing a photosensitive substance; and the preparation method of the three-component solid reagents comprises: respectively diluting the R1 reagent, the R2 reagent and the R3 reagent with a diluent according to a certain concentration and then freeze-drying them.

[0007] As a further improvement, in the R1 reagent, the coating ratio of the latex microspheres containing fluorescent and dye substances to the progesterone antibody is 1 mg: 2.5-30 μg.

[0008] As a further improvement, in the R2 reagent, the labeling ratio of the water-soluble biotin derivative to the progesterone peptide is 5 mol:1 mol.

[0009] As a further improvement, in the R3 reagent, the coating ratio of the latex microspheres containing the photosensitive substance to streptavidin is 1 mg:50 μg.

[0010] As a further improvement, the mass ratio of the progesterone peptide in the R2 reagent to the progesterone in the R1 reagent is 16.67-50:1.

[0011] As a further improvement, the molar ratio of the water-soluble biotin derivative in the R2 reagent to the streptavidin in the R3 reagent is not greater than 5:1 and not less than 4:1.

[0012] As a further improvement, the actual working contents of the progesterone antibody (PROG-Ab) in the R1 reagent, the progesterone peptide (PROGAg-BSA) in the R2 reagent and the streptavidin (SA) in the R3 reagent after dilution with the diluent are 5-15 ng / person, 100-500 ng / person and 250-300 ng / person respectively.

[0013] As a further improvement, the diluent is two of H1 buffer, H2 buffer or H3 buffer; the components of the H1 buffer are: 50mM-250mM (w / v) TRIS, 0.1%-1.0% (w / v) NaCl, 0.1%-1.0% (w / v) Tween20, 0.1%-1.0% (v / v) Proclin300, 0.1%-5.0% (w / v) bovine serum albumin, pH 6.8-7.4; the components of the H2 buffer are: 50mM-250mM (w / v) MES, 0.1%-1.0% (w / v) NaCl, 0.1%-1.0% (w / v) Tween20, 0.1%-1.0% (v / v) Proclin300, 0.1%-5.0% (w / v) bovine serum albumin, pH 6.0-8.0; the components of the H3 buffer are: 100mM-500mM (w / v) citric acid, 100mM-500mM (w / v) sodium citrate, 0.1%-1.0% (w / v) NaCl, 0.1%-1.0% (w / v) Tween20, 0.1%-1.0% (v / v) Proclin300, 0.1%-5.0% (w / v) bovine serum albumin, pH 4.0-5.0.

[0014] A preparation method of the above-mentioned kit comprises: diluting the R1 reagent, the R2 reagent and the R3 reagent with a diluent according to a certain concentration and then freeze-drying them. As a further improvement, the freeze-drying process of the freeze-drying is: pre-freezing at -50°C for 2h, vacuuming at -45°C for 1h, freeze-drying at -40°C for 1h, freeze-drying at -35°C for 8h, freeze-drying at -30°C for 2h, freeze-drying at -20°C for 2h, freeze-drying at -10°C for 2h, freeze-drying at 0°C for 4h, freeze-drying at 10°C for 2h, freeze-drying at 20°C for 2h, freeze-drying at 30°C for 2h and heat preservation.

[0015] An application method of the above-mentioned kit includes: diluting the sample with a sample diluent, adding the three-component solid reagent in the kit, and obtaining the test result after the three-component solid reagent is re-dissolved and reacting for a period of time.

[0016] The beneficial effects of the present invention are:

[0017] The progesterone detection kit of the present invention comprises three-component solid reagents, wherein the R1 reagent and the R2 reagent are combined together due to the antigen-antibody reaction between the progesterone antibody and the progesterone peptide, and the R2 reagent and the R3 reagent are combined together due to the affinity reaction between the biotin derivative and streptavidin, that is, the R1 reagent, the R2 reagent and the R3 reagent are combined together. The photosensitive material in the R3 reagent receives an excitation light of a certain wavelength and transfers energy to excite the oxygen in the environment into singlet oxygen, and further excites fluorescence with the fluorescence and dye material in the R1 reagent. When the sample is added, after synchronous re-dissolution, the progesterone in the sample and the progesterone peptide in the R2 reagent synchronously compete for the progesterone antibody in the R1 reagent, so that the binding degree of the progesterone antibody in the R1 reagent and the progesterone peptide in the R2 reagent is reduced, and the measured fluorescence intensity decreases, thereby realizing the quantitative detection of the content of progesterone in the sample according to the size of the fluorescence intensity. This detection method only requires mixing the sample and the sample diluent and then adding them to a three-component solid reagent, and then testing after the three-component solid reagent and the sample react synchronously. There is no sequence of reactions and washing steps in the traditional competitive method, and no dissociation agent is required. The operation is simple and convenient for the detection of progesterone.

[0018] The detection coefficient of variation CV of the progesterone detection kit of the present invention is less than or equal to 5%, and the linear correlation r with the progesterone detection kit (electrochemiluminescence method) of Roche Company reaches 0.9984.

[0019] The progesterone detection kit of the present invention has strong detection specificity and high sensitivity, and the reaction time only requires 15 minutes. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 The solid reagent of the kit of Example 12 provided in Example 13 of the present invention is placed at 2-8°C for 10 days, and then the three-component reagent is simultaneously reconstituted to perform a linear fitting graph on the RLU data obtained from testing ≥40 clinical samples.

[0022] Figure 2 The solid reagent of the kit of Example 12 provided in Example 13 of the present invention is placed at 45° C. for 10 days, and then the three-component reagent is simultaneously reconstituted to perform a linear fitting graph on the RLU data obtained from testing ≥40 clinical samples. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention claimed for protection, but merely represents the selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0024] The embodiment of the present invention provides a progesterone detection kit, including three-component solid reagents; the kit can be made into a reagent card, which can intercept red blood cells and impurities. The three-component solid reagent includes R1 reagent, R2 reagent and R3 reagent; the R1 reagent is a progesterone antibody coated with latex microspheres containing fluorescent and dye substances; the R2 reagent is a progesterone peptide modified with a water-soluble biotin derivative; the R3 reagent is streptavidin coated with latex microspheres containing a photosensitive substance; the preparation method of the three-component solid reagent is: the R1 reagent, the R2 reagent and the R3 reagent are diluted with a diluent according to a certain concentration and then freeze-dried. The R1 reagent and the R2 reagent are combined together due to the antigen-antibody reaction between the progesterone antibody and the progesterone peptide, and the R2 reagent and the R3 reagent are combined together due to the affinity reaction between the biotin derivative and the streptavidin, that is, the R1 reagent, the R2 reagent and the R3 reagent are combined together. The photosensitive substance in the R3 reagent receives excitation light of a certain wavelength and transfers energy to excite oxygen in the environment into singlet oxygen, which is further excited to produce fluorescence by reacting with the fluorescent and dye substances in the R1 reagent.

[0025] In some embodiments, the fluorescent and dye substances are thiophene derivatives and europium chelates, and the photosensitive substance is a phthalocyanine photosensitizer.

[0026] In some embodiments, the latex microspheres are carboxylated latex microspheres, such as carboxylated polystyrene microspheres.

[0027] In some embodiments, the progesterone antibody (PROG-Ab) is selected from one of Cat: V-09201-1 (Nanjing GenScript), Cat: R196n6 (Nanjing Oukai), and Cat: A208 (EastCoast Bio).

[0028] In some embodiments, the water-soluble biotin derivative (Bio) is a water-soluble biotin ester, selected from one of Cat: QBD10198 (sigma), Cat: B419512 (Shanghai Aladdin Biochemical Technology Co., Ltd.), and Cat: 21338 (ThermoFisher).

[0029] In some embodiments, the progesterone peptide (PROG Ag-BSA) is selected from one of Cat: HP05S111 (Beijing Keyue Zhongkai), Cat: V56050369 (Biospacific), and Cat: HF-036502 (Shanghai Hanzun).

[0030] In some embodiments, the streptavidin (SA) is selected from one of Cat: S4762 (sigma), Cat: S817490 (McLean), and Cat: S274235 (Shanghai Aladdin Biochemical Technology Co., Ltd.).

[0031] In some embodiments, in the R1 reagent, the coating ratio of the latex microspheres containing fluorescent and dye substances to the progesterone antibody (PROG-Ab) is 1 mg: 2.5-30 μg. Within this range, the correlation of the progesterone test reaction is better.

[0032] In some embodiments, in the R2 reagent, the labeling ratio of the water-soluble biotin derivative (Bio) to the progesterone peptide (PROG Ag-BSA) is 5 mol: 1 mol. Within this range, the progesterone test reaction correlation is better.

[0033] In some embodiments, in the R3 reagent, the coating ratio of the latex microspheres containing the photosensitive substance to streptavidin (SA) is 1 mg:50 μg. Within this range, the correlation of the progesterone test reaction is better.

[0034] In some embodiments, the mass ratio of the progesterone peptide (PROG Ag-BSA) in the R2 reagent to the progesterone antibody (PROG-Ab) in the R1 reagent is 16.67-50:1. At this ratio, the progesterone peptide in the R2 reagent has a good competitive relationship with the progesterone in the sample to be tested.

[0035] In some embodiments, the molar ratio of the water-soluble biotin derivative (Bio) in the R2 reagent to the streptavidin (SA) in the R3 reagent is not greater than 5:1 and not less than 4:1. At this ratio, the affinity reaction between the water-soluble biotin derivative (Bio) and streptavidin (SA) is optimal.

[0036] In some embodiments, the diluent is two of H1 buffer, H2 buffer or H3 buffer; the components of the H1 buffer are: 50mM-250mM (w / v) TRIS, 0.1%-1.0% (w / v) NaCl, 0.1%-1.0% (w / v) Tween20, 0.1%-1.0% (v / v) Proclin300, 0.1%-5.0% (w / v) bovine serum albumin, pH 6.8-7.4; the components of the H2 buffer are: 50mM-250mM (w / v) MES, 0.1%-1.0% (w / v) NaCl, 0.1%-1.0% (w / v) Tween20, 0.1%-1.0% (v / v) Proclin300, 0.1%-5.0% (w / v) bovine serum albumin, pH 6.0-8.0; the components of the H3 buffer are: 100mM-500mM (w / v) citric acid, 100mM-500mM (w / v) sodium citrate, 0.1%-1.0% (w / v) NaCl, 0.1%-1.0% (w / v) Tween20, 0.1%-1.0% (v / v) Proclin300, 0.1%-5.0% (w / v) bovine serum albumin, pH 4.0-5.0.

[0037] In some embodiments, the actual working contents of the progesterone antibody (PROG-Ab) in the R1 reagent, the progesterone peptide (PROG Ag-BSA) in the R2 reagent, and the streptavidin (SA) in the R3 reagent after dilution with the diluent are 5-15 ng / person, 100-500 ng / person, and 250-300 ng / person, respectively.

[0038] A preparation method of the above-mentioned kit comprises: diluting the R1 reagent, the R2 reagent and the R3 reagent with a diluent according to a certain concentration and then freeze-drying them. In some embodiments, the freeze-drying process of the freeze-drying is: pre-freezing at -50°C for 2h, vacuuming at -45°C for 1h, freeze-drying at -40°C for 1h, freeze-drying at -35°C for 8h, freeze-drying at -30°C for 2h, freeze-drying at -20°C for 2h, freeze-drying at -10°C for 2h, freeze-drying at 0°C for 4h, freeze-drying at 10°C for 2h, freeze-drying at 20°C for 2h, freeze-drying at 30°C for 2h and heat preservation. Freeze-drying under these conditions has a better effect, and the freeze-dried samples are easy to carry.

[0039] An application method of the above-mentioned kit includes: diluting the sample with a sample diluent, adding the three-component solid reagent in the kit, and then re-dissolving the three-component solid reagent in the sample diluent, and reacting for a period of time to obtain the test result. During the test, the progesterone in the sample and the progesterone peptide in the R2 reagent simultaneously compete for the progesterone antibody in the R1 reagent, so that the combination of the R1 reagent and the R2 reagent is reduced, and then the combination of the fluorescence and the dye substance and the photosensitive substance is reduced, thereby reducing the fluorescence intensity. The higher the progesterone content in the sample, the lower the fluorescence intensity. Detect the fluorescence intensity, and then evaluate the progesterone content in the sample according to the size of the fluorescence intensity, so as to achieve the detection of the progesterone content. This detection method only needs to re-dissolve the three-component solid reagent and the sample synchronously and then conduct the test. There is no sequence of reactions and cleaning steps in the traditional competition method, and no dissociation agent is required. The operation is simple and convenient for the detection of progesterone.

[0040] In some embodiments, the sample is selected from an antigen, serum, plasma, or whole blood.

[0041] In some embodiments, the sample diluent is one of purified water, 0.9% (w / v) NaCl solution, and 50mM TBST (pH 7.4) buffer, wherein the composition of 50mM TBST pH 7.4 buffer is 0.05M (w / v) Tris + 1.5% (w / v) BSA + 0.9% (w / v) NaCl + 0.2% (w / v) TW-20 + 0.1% (v / v) ProClin 300.

[0042] Example 1

[0043] Preparation of a progesterone detection kit

[0044] The latex microspheres containing fluorescent and dye substances were coated with progesterone antibodies, and the initial coating ratio was 1 mg:50 μg of latex microspheres containing fluorescent and dye substances: progesterone antibodies to prepare R1 reagent. The coating method was as follows: the latex microspheres containing fluorescent and dye substances were washed twice with activation coupling buffer (50 mM (w / v) MES pH 5.0), the latex microspheres containing fluorescent and dye substances were mixed with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), activated in activation coupling buffer at 37°C for 10 minutes, and then a certain amount of progesterone antibodies (PROG-Ab) were added to react at 37°C for 3 hours, and then BSA was used to block at 37°C for 0.5 hours. After the blocking was completed, H1 buffer was replaced for washing, centrifugation and storage, and the storage concentration was 5 mg / mL.

[0045] The water-soluble biotin derivative was used to label the progesterone peptide, and the initial labeling ratio was 5 mol:1 mol of the water-soluble biotin derivative:progesterone peptide to prepare the R2 reagent. The labeling method was as follows: the water-soluble biotin derivative (Bio) and progesterone peptide (PROG Ag-BSA) required for labeling were added to the labeling buffer (10 mM (w / v) Na2HPO4·12H2O+NaH2PO4·2H2OpH7.4), reacted at room temperature for 1 hour, and then glycine was added for blocking for 0.5 hours. After the blocking was completed, the unreacted water-soluble biotin derivative was removed by dialysis through a dialysis bag, and then the protein concentration was determined by a micro-spectrophotometer. The concentration of 1 mg / mL was qualified.

[0046] The latex microspheres containing the photosensitive substance were coated with streptavidin, and the coating ratios were 1 mg:30 μg, 1 mg:50 μg, and 1 mg:100 μg of latex microspheres containing the photosensitive substance: streptavidin, respectively, to prepare the R3 reagent. The coating method was as follows: the latex microspheres containing the phthalocyanine photosensitizer were washed twice with an activated coupling buffer (50 mM (w / v) MES pH 5.0), the latex microspheres containing the phthalocyanine photosensitizer were reacted with streptavidin (SA) and EDC in the coupling buffer at 37°C for 2 h, and then blocked with BSA at 37°C for 0.5 h. After the blocking, the H1 buffer was replaced for washing, centrifugation, and storage, and the storage concentration was 5 mg / mL.

[0047] Take R1 reagent, R2 reagent, and R3 reagent, dilute them with H1 buffer, the actual working content of progesterone antibody (PROG-Ab) in the diluted R1 reagent is 250ng / person, the actual working content of progesterone peptide (PROG Ag-BSA) in the R2 reagent is 250ng / person, and the actual working content of streptavidin (SA) in the R3 reagent is 75ng / person, 125ng / person, and 250ng / person, respectively, freeze-dried into three-component solid reagents, and packaged into a test kit. The freeze-drying process is: pre-freezing at -50°C for 2h, vacuuming at -45°C for 1h, freeze-drying at -40°C for 1h, freeze-drying at -35°C for 8h, freeze-drying at -30°C for 2h, freeze-drying at -20°C for 2h, freeze-drying at -10°C for 2h, freeze-drying at 0°C for 4h, freeze-drying at 10°C for 2h, freeze-drying at 20°C for 2h, freeze-drying at 30°C for 2h and heat preservation.

[0048] The components of the H1 buffer are: 250mM (w / v) TRIS, 0.9% (w / v) NaCl, 1.0% (w / v) Tween20, 0.5% (v / v) Proclin300, 5% (w / v) bovine serum albumin, pH 7.4; the components of the H3 buffer are: 100mM (w / v) citric acid, 100mM (w / v) sodium citrate, 0.9% (w / v) NaCl, 1.0% (w / v) Tween20, 0.5% (v / v) Proclin300, 5% (w / v) bovine serum albumin, pH 5.0.

[0049] Example 2

[0050] The coating ratio of R1 reagent is 1 mg:50 μg, and the actual working content of the diluted progesterone antibody (PROG-Ab) is 250 ng / person; the labeling ratio of R2 reagent is 5 mol:1 mol, and the actual working content of the diluted progesterone peptide (PROG Ag-BSA) is 250 ng / person; the coating ratios of R3 reagent are 1 mg:30 μg, 1 mg:50 μg, and 1 mg:100 μg, respectively, and the actual working content of the diluted streptavidin (SA) is 150 ng / person, 250 ng / person, and 500 ng / person, respectively; other operations are the same as Example 1.

[0051] Example 3

[0052] The coating ratio of R1 reagent is 1 mg:50 μg, and the actual working content of the diluted progesterone antibody (PROG-Ab) is 250 ng / person; the labeling ratio of R2 reagent is 5 mol:1 mol, and the actual working content of the diluted progesterone peptide (PROG Ag-BSA) is 250 ng / person; the coating ratio of R3 reagent is 1 mg:30 μg, 1 mg:50 μg, 1 mg:100 μg, and the actual working content of the diluted streptavidin (SA) is 300 ng / person, 500 ng / person, and 1000 ng / person, respectively; other operations are the same as Example 1.

[0053] Example 4

[0054] The coating ratios of the R1 reagent were 1 mg:2.5 μg, 1 mg:5 μg, 1 mg:10 μg, 1 mg:30 μg, and 1 mg:50 μg, respectively, and the actual working contents of the progesterone antibody (PROG-Ab) after dilution were 250 ng / person, 150 ng / person, 50 ng / person, 25 ng / person, and 12.5 ng / person, respectively; the initial labeling ratio of the R2 reagent was 5 mol:1 mol, and the actual working content of the progesterone peptide (PROG Ag-BSA) after dilution was 250 ng / person; the coating ratio of the R3 reagent was 1 mg:50 μg, and the actual working content of the streptavidin (SA) after dilution was 250 ng / person; other operations were the same as in Example 1.

[0055] Example 5

[0056] The coating ratios of the R1 reagent were 1 mg:2.5 μg, 1 mg:5 μg, 1 mg:10 μg, 1 mg:30 μg, and 1 mg:50 μg, respectively, and the actual working contents of the progesterone antibody (PROG-Ab) after dilution were 75 ng / person, 45 ng / person, 15 ng / person, 7.5 ng / person, and 3.75 ng / person, respectively; the initial labeling ratio of the R2 reagent was 5 mol:1 mol, and the actual working content of the progesterone peptide (PROG Ag-BSA) after dilution was 250 ng / person; the coating ratio of the R3 reagent was 1 mg:50 μg, and the actual working content of the streptavidin (SA) after dilution was 250 ng / person; other operations were the same as in Example 1.

[0057] Example 6

[0058] The coating ratios of the R1 reagent were 1 mg:2.5 μg, 1 mg:5 μg, 1 mg:10 μg, 1 mg:30 μg, and 1 mg:50 μg, respectively, and the actual working contents of the progesterone antibody (PROG-Ab) after dilution were 25 ng / person, 15 ng / person, 5 ng / person, 2.5 ng / person, and 1.25 ng / person, respectively; the labeling ratio of the R2 initial reagent was 5 mol:1 mol, and the actual working content of the progesterone peptide (PROG Ag-BSA) after dilution was 250 ng / person; the coating ratio of the R3 reagent was 1 mg:50 μg, and the actual working content of the streptavidin (SA) after dilution was 250 ng / person; other operations were the same as in Example 1.

[0059] Example 7

[0060] The coating ratios of the R1 reagent were 1 mg:5 μg, and the actual working content of the diluted progesterone antibody (PROG-Ab) was 7.5 ng / person; the labeling ratios of the R2 reagent were 20 mol:1 mol, 10 mol:1 mol, 5 mol:1 mol, and 2.5 mol:1 mol, and the actual working content of the diluted progesterone peptide (PROG Ag-BSA) was 500 ng / person; the coating ratio of the R3 reagent was 1 mg:50 μg, and the actual working content of the diluted streptavidin (SA) was 250 ng / person; other operations were the same as in Example 1.

[0061] Example 8

[0062] The coating ratios of the R1 reagent were 1 mg:5 μg, and the actual working content of the diluted progesterone antibody (PROG-Ab) was 7.5 ng / person; the labeling ratios of the R2 reagent were 20 mol:1 mol, 10 mol:1 mol, 5 mol:1 mol, and 2.5 mol:1 mol, and the actual working content of the diluted progesterone peptide (PROG Ag-BSA) was 250 ng / person; the coating ratio of the R3 reagent was 1 mg:50 μg, and the actual working content of the diluted streptavidin (SA) was 250 ng / person; other operations were the same as in Example 1.

[0063] Example 9

[0064] The coating ratios of the R1 reagent were 1 mg:5 μg, and the actual working content of the diluted progesterone antibody (PROG-Ab) was 7.5 ng / person; the labeling ratios of the R2 reagent were 20 mol:1 mol, 10 mol:1 mol, 5 mol:1 mol, and 2.5 mol:1 mol, and the actual working content of the diluted progesterone peptide (PROG Ag-BSA) was 100 ng / person; the coating ratio of the R3 reagent was 1 mg:50 μg, and the actual working content of the diluted streptavidin (SA) was 250 ng / person; other operations were the same as in Example 1.

[0065] Comparative Example 1

[0066] The initial coating ratio of R1 reagent is 1 mg:50 μg, and the actual working content of the diluted progesterone antibody (PROG-Ab) is 250 ng / person; the initial labeling ratio of R2 reagent is 5 mol:1 mol, and the actual working content of the diluted progesterone peptide (PROG Ag-BSA) is 250 ng / person; the coating ratios of R3 reagent are 1 mg:30 μg, 1 mg:50 μg, and 1 mg:100 μg, respectively, and the actual working contents of streptavidin (SA) after dilution are 37.5 ng / person, 62.5 ng / person, and 125 ng / person, respectively; other operations are the same as Example 1.

[0067] Comparative Example 2

[0068] The coating ratios of the R1 reagent were 1 mg:2.5 μg, 1 mg:5 μg, 1 mg:10 μg, 1 mg:30 μg, and 1 mg:50 μg, respectively, and the actual working contents of the progesterone antibody (PROG-Ab) after dilution were 500 ng / person, 300 ng / person, 100 ng / person, 50 ng / person, and 25 ng / person, respectively; the initial labeling ratio of the R2 reagent was 5 mol:1 mol, and the actual working content of the progesterone peptide (PROG Ag-BSA) after dilution was 250 ng / person; the coating ratio of the R3 reagent was 1 mg:50 μg, and the actual working content of the streptavidin (SA) after dilution was 250 ng / person; other operations were the same as in Example 1.

[0069] Comparative Example 3

[0070] The coating ratios of the R1 reagent were 1 mg:5 μg, and the actual working content of the diluted progesterone antibody (PROG-Ab) was 7.5 ng / person; the labeling ratios of the R2 reagent were 20 mol:1 mol, 10 mol:1 mol, 5 mol:1 mol, and 2.5 mol:1 mol, and the actual working content of the diluted progesterone peptide (PROG Ag-BSA) was 50 ng / person; the coating ratio of the R3 reagent was 1 mg:50 μg, and the actual working content of the diluted streptavidin (SA) was 250 ng / person; other operations were the same as in Example 1.

[0071] The test kits of Example 1-9 and Comparative Example 1-3 were used to test the samples. The test method was as follows: the sample was diluted with a sample diluent, the dilution concentration was 1:15, and then the three-component solid reagent in the test kit was added. After the three-component solid reagent was re-dissolved, the relative light unit RLU was tested after a period of reaction. The test results are shown in Table 1-2 to Table 12-2, and the sample gradient is calculated as shown in Table 1-3 to Table 12-3. The composition of the sample diluent is 0.9% (w / v) NaCl + 0.1% (v / v) ProClin 300.

[0072] Table 1-1 Labeling coating ratio of R1, R2, and R3 reagents in Example 1 and actual working content after dilution

[0073]

[0074] Note: Taking Example 1 and R3 coating ratio of 1 mg:30 μg as an example, the content of PROG-Ab after reconstitution of one dose of R1 reagent in this example is 250 ng, the content of PROG-Ag-BSA after reconstitution of one dose of R2 reagent is 250 ng, and the content of SA after reconstitution of one dose of R3 reagent is 75 ng. The same applies to the following examples and comparative examples.

[0075] m(Ag-BSA) / m(Ab)=actual working content of PROG-Ag-BSA / actual working content of PROG-Ab

[0076] =250ng / 250ng=1, the following examples and comparative examples are calculated in the same way.

[0077] n(Bio):n(SA)=(content of PROG-Ag-BSA in the actual working solution / molecular weight of PROG-Ag-BSA*the molar ratio of Bio) / (content of SA in the actual working solution / molecular weight of SA)=(250ng / 60kD*5) / (75ng / 60kD)=17, the molecular weight of PROG-Ag-BSA is about 60kD, the molecular weight of SA is about 60kD, and the calculation of the following examples and comparative examples is the same.

[0078] Table 1-2 Example 1 Test clinical sample RLU data

[0079]

[0080]

[0081] Table 1-3 Example 1 Test clinical sample RLU multiple ratio (low concentration: adjacent high concentration)

[0082]

[0083] Table 2-1 R1, R2, R3 reagent labeling coating ratio and actual working content after dilution in Example 2

[0084]

[0085] Table 2-2 Example 2 Test clinical sample RLU data

[0086]

[0087] Table 2-3 Example 2 Test clinical sample RLU multiple ratio (low concentration: adjacent high concentration)

[0088]

[0089]

[0090] Table 3-1 Labeling coating ratio of R1, R2, and R3 reagents in Example 3 and actual working content after dilution

[0091]

[0092] Table 3-2 Example 3 Test Clinical Sample RLU Data

[0093]

[0094] Table 3-3 Example 3 Test clinical sample RLU multiple ratio (low concentration: adjacent high concentration)

[0095]

[0096] Table 4-1 Labeling coating ratio of R1, R2, and R3 reagents in comparative example 1 and actual working content after dilution

[0097]

[0098]

[0099] Table 4-2 RLU data of clinical samples tested in comparative example 1

[0100]

[0101] Table 4-3 RLU ratio of clinical samples tested in comparative example 1 (low concentration: adjacent high concentration)

[0102]

[0103] As shown in Table 1-1 to Table 4-3, the RLU value of the clinical sample is related to the increase or decrease of the photosensitizer in the reaction system and the binding amount of SA and Bio. Under the conditions that the actual working contents of PROG-Ab and PROG-Ag-BSA are 250ng and 250ng respectively, too high or too low streptavidin content may cause no distinction or small distinction in the low value segment of the progesterone sample test, and too large distinction in the medium and high value segments.

[0104] Since there is a certain ratio for the affinity reaction between streptavidin (SA) and the water-soluble biotin derivative (Bio) (1 mol of streptavidin (SA) binds to 4 mol of biotin derivative ester (Bio)), based on the experimental data of Examples 1-3 and Comparative Example 1, n(Bio):n(SA) should not be greater than 5, and n(Bio):n(SA) should not be less than 4.

[0105] According to the data of Examples 1-3 and Comparative Example 1, the present invention determines that the coating ratio of R3 reagent is 1 mg:50 μg, and the actual working content of SA in R3 reagent is 250 ng. At this time, n(Bio):n(SA)=5 mol:1 mol. Under this condition, the low value has a subtle distinction and the distinction between medium and high values ​​tends to approach the RLU distinction of Roche test.

[0106] Table 5-1 Labeling coating ratio of R1, R2, and R3 reagents in Example 4 and actual working content after dilution

[0107]

[0108] Table 5-2 Example 4 Test Clinical Sample RLU Data

[0109]

[0110] Table 5-3 Example 4 Test Clinical Sample RLU Ratio (Low Concentration: Adjacent High Concentration)

[0111]

[0112] Table 6-1 Labeling coating ratio of R1, R2, and R3 reagents in Example 5 and actual working content after dilution

[0113]

[0114]

[0115] Table 6-2 Example 5 Test Clinical Sample RLU Data

[0116]

[0117] Table 6-3 Example 5 Test Clinical Sample RLU Ratio (Low Concentration: Adjacent High Concentration)

[0118]

[0119] Table 7-1 Labeling coating ratio of R1, R2, and R3 reagents in Example 6 and actual working content after dilution

[0120]

[0121] Table 7-2 Example 6 Test Clinical Sample RLU Data

[0122]

[0123] Table 7-3 Example 6 Test Clinical Sample RLU Ratio (Low Concentration: Adjacent High Concentration)

[0124]

[0125] Table 8-1 Labeling coating ratio of R1, R2, and R3 reagents in comparative example 2 and actual working content after dilution

[0126]

[0127] Table 8-2 RLU data of clinical samples tested in comparative example 2

[0128]

[0129]

[0130] Table 8-3 RLU ratio of clinical samples tested in comparative example 2 (low concentration: adjacent high concentration)

[0131]

[0132] As shown in Table 5-1-Table 8-3, the RLU value of the clinical sample is related to the actual working content of PROG-Ab in the R1 reagent and the increase or decrease of the fluorescent and dye substances in the reaction system. When n(Bio):n(SA)=5:1, when m(Ag-BSA) / m(Ab)=16.67-50:1, the RLU multiple of the progesterone sample is larger in the low value segment and the medium and high value segment is closer to the RLU multiple of Roche assay; under the conditions that the actual working contents of SA and PROG-Ag-BSA are 250ng and 250ng respectively, too high or too low PROG-Ab content may cause no distinction or small distinction in the low value segment of the progesterone sample test, and too large distinction in the medium and high value segments.

[0133] According to the data of Examples 4-6 and Comparative Example 2, the present invention determines that the coating ratio of R1 reagent is 1 mg:5 μg, and the actual working content of PROG-Ab in R1 reagent is 7.5 ng. At this time, m(Ag-BSA) / m(Ab)=33.3:1. Under this condition, the low value has a good distinction and the distinction between medium and high values ​​tends to approach the RLU distinction of Roche test.

[0134] Due to the different affinities between different antibodies and progesterone peptides, the competitive abilities of progesterone peptides with the progesterone samples to be tested are different, and researchers need to try different ratios of conditions. The present invention only provides a development method. In the present invention, it is found that when the ratio of progesterone peptide to antibody is 16.67-50:1, progesterone peptide has a good competitive relationship with the progesterone sample to be tested.

[0135] Table 9-1 Labeling coating ratio of R1, R2, and R3 reagents in Example 7 and actual working content after dilution

[0136]

[0137] Table 9-2 RLU data of clinical samples tested in Example 7

[0138]

[0139] Table 9-3 RLU data of clinical samples tested in Example 7

[0140]

[0141] Table 10-1 Labeling coating ratio of R1, R2, and R3 reagents in Example 8 and actual working content after dilution

[0142]

[0143] Table 10-2 RLU data of clinical samples tested in Example 8

[0144]

[0145]

[0146] Table 10-3 RLU data of clinical samples tested in Example 8

[0147]

[0148] Table 11-1 Labeling coating ratio of R1, R2, and R3 reagents in Example 9 and actual working content after dilution

[0149]

[0150] Table 11-2 RLU data of clinical samples tested in Example 9

[0151]

[0152] Table 11-3 RLU data of clinical samples tested in Example 9

[0153]

[0154]

[0155] Table 12-1 Labeling coating ratio of R1, R2, and R3 reagents in comparative example 3 and actual working content after dilution

[0156]

[0157] Table 12-2 RLU data of clinical samples tested in comparative example 3

[0158]

[0159] Table 12-3 RLU data of clinical samples tested in comparative example 3

[0160]

[0161] As shown in Table 9-1 to Table 12-3, as the labeling ratio of R2 reagent increases, the RLU of the progesterone sample tested tends to decrease; as the actual working content of PROG-Ag-BSA in R2 reagent increases, the RLU of the progesterone sample tested tends to increase first and then decrease. Since the theoretical molar ratio of streptavidin and water-soluble biotin derivative is 1:4, the high-labeled water-soluble biotin derivative may be excessive, resulting in some R2 reagents binding to R1 reagents, but not to R3 reagents. Therefore, the RLU of the progesterone sample tested decreases as the labeling ratio of R2 reagent increases, and after the actual working content of PROG-Ag-BSA in R2 reagent increases to a certain amount, the RLU of the test decreases.

[0162] According to the experimental data of Examples 7-9 and Comparative Example 3, the present invention determines that the labeling ratio of R2 reagent is 5 mol: 1 mol, and the actual working content of PROG-Ag-BSA in R2 reagent is 250 ng. At this time, m(Ag-BSA) / m(Ab)=33.3:1 and n(Bio):n(SA)=5:1. Under this ratio condition, the low value has a good distinction and the distinction between medium and high values ​​has a trend close to the RLU distinction of Roche test. And according to the experimental data of Examples 7-9 and Comparative Example 3, the experimental results of Examples 1-6 and Comparative Example 1-2 are verified again. When the ratio of each substance is m(Ag-BSA) / m(Ab)=16.67-50:1 and n(Bio):n(SA) is not more than 5:1 and not less than 4:1, there is a good distinction between low values ​​and a distinction between medium and high values ​​close to Roche.

[0163] Example 10

[0164] The coating ratio of R1 reagent is 1 mg:5 μg; the labeling ratio of R2 reagent is 5 mol:1 mol; the coating ratio of R3 reagent is 1 mg:50 μg; the formula of the buffer solution and the actual working content after dilution are shown in Table 13, and other operations are the same as Example 1.

[0165] Table 13 Example 10 Buffer formulation and actual working content after dilution

[0166] H1 Buffer content TRIS 250mM NaCl 0.9％ Tw20 1.0％ Proclin300 0.5％ BSA 5.0％ pH 7.4 The actual working content of R1 reagent after dilution 7.5ng The actual working content of R2 reagent after dilution 250ng Actual working content of R3 reagent after dilution 250ng

[0167] Embodiment 11

[0168] The coating ratio of R1 reagent is 1 mg:5 μg; the labeling ratio of R2 reagent is 5 mol:1 mol; the coating ratio of R3 reagent is 1 mg:50 μg; the formula of the buffer solution and the actual working content after dilution are shown in Table 14, and other operations are the same as Example 1.

[0169] Table 14 Example 11 Buffer formula and actual working content after dilution

[0170]

[0171]

[0172] Example 12

[0173] The coating ratio of R1 reagent is 1 mg:5 μg; the labeling ratio of R2 reagent is 5 mol:1 mol; the coating ratio of R3 reagent is 1 mg:50 μg; R1 reagent and R3 reagent are diluted with H1 buffer, and R2 reagent is diluted with H2 buffer. The formula of the buffer and the actual working content after dilution are shown in Table 15, and other operations are the same as in Example 1.

[0174] Table 15 Example 8 Buffer Formula and Actual Working Content after Dilution

[0175] H1 Buffer content H3 Buffer content TRIS 250mM Citric Acid 200mM Sodium Citrate 300mM NaCl 0.9％ NaCl 0.9％ Tw20 1.0％ Tw20 1.0％ Proclin300 0.5％ Proclin300 0.5％ BSA 5.0％ BSA 5.0％ pH 7.4 pH 5.5 R1 Reagent 7.5ng R2 Reagent 250ng R3 Reagent 250ng

[0176] Comparative Example 4

[0177] The coating ratio of R1 reagent is 1 mg:5 μg; the labeling ratio of R2 reagent is 5 mol:1 mol; the coating ratio of R3 reagent is 1 mg:50 μg; the formula of the buffer solution and the actual working content after dilution are shown in Table 16, and other operations are the same as Example 1.

[0178] Table 16 Comparative Example 4 Buffer Formula and Actual Working Content after Dilution

[0179] H2 Buffer content MES 250mM NaCl 0.9％ Tw20 1.0％ Proclin300 0.5％ BSA 5.0％ pH 6.0 R1 Reagent 7.5ng R2 Reagent 250ng R3 Reagent 250ng

[0180] The samples were tested using the kits of Examples 10-11 and Comparative Example 4, and the test results are shown in Tables 17-21.

[0181] Table 17 Fitting concentrations of the kit tests of Examples 10-12 and Comparative Example 4

[0182]

[0183]

[0184] Table 18 CV and range of test samples of the kit of comparative example 4

[0185]

[0186] Table 19 CV and range of test samples of the kit of Example 10

[0187]

[0188] Table 20 CV and range of test samples of the kit of Example 11

[0189]

[0190] Table 21 CV and range of test samples of the kit of Example 12

[0191]

[0192] It can be seen from Table 17 that the reagent fitting concentration diluted using the composite formula of H1 buffer and H3 buffer has the best deviation from the Roche sample concentration.

[0193] As can be seen from Table 18, the reagent diluted with H2 buffer was lyophilized and then tested for the progesterone sample to obtain 5 parallel tests at the same concentration. The CV and range were both large, which had a large error on the final result.

[0194] As shown in Table 19, the reagent diluted with H1 buffer was lyophilized and tested for progesterone samples, and the CVs of 5 parallel tests at the same concentration were all within 10%, but the RLU range of some concentration values ​​was large, which had a large error on the final result.

[0195] As shown in Table 20, the reagent diluted with H3 buffer was lyophilized and tested for progesterone samples, and the CVs of 5 parallel tests at the same concentration were all within 10%, but the RLU range of some concentration values ​​was large, which had a large error on the final result.

[0196] As can be seen from Table 21, the reagent diluted with the composite formula of H1 buffer and H3 buffer was freeze-dried and tested for the progesterone sample to obtain CVs of 5 parallel tests at the same concentration within 5%, and the range of each concentration was within 10%, which had a small impact on the final error result.

[0197] Embodiment 13

[0198] The solid reagent of the kit of the preferred embodiment 12 was placed at 2-8°C and 45°C for 10 days, and then the three-component reagent was simultaneously reconstituted to test ≥40 clinical samples. The obtained data were linearly fitted to obtain Table 22, Table 23 and Figure 1 , Figure 2 .

[0199] The synchronous reconstitution method is: dilute the sample with sample diluent, the dilution concentration is sample: diluent is 1:15, then add the three-component solid reagent in the kit, wait for the three-component solid reagent to be reconstituted, test the relative light unit RLU after a period of reaction, and calculate the progesterone concentration. The sample diluent is 0.9% NaCl solution.

[0200] Table 22 The solid reagents of the kit of preferred embodiment 12 were placed at 2-8°C for 10 days and then tested on ≥40 clinical samples

[0201]

[0202]

[0203] Table 23 Solid reagents of the kit of preferred embodiment 8 were placed at 45°C for 10 days and then tested on ≥40 clinical samples

[0204]

[0205]

[0206] From Tables 22, 23, Figure 1-2It can be seen that the three-component solid reagents of the kit of preferred embodiment 12 are tested by simultaneous reconstitution, and the acceleration at 45° C. has no effect on the correlation r of the sample concentration with the Roche assay.

[0207] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A progesterone detection kit, characterized in that: The invention comprises a three-component solid reagent, wherein the three-component solid reagent comprises R1 reagent, R2 reagent and R3 reagent; the R1 reagent is a progesterone antibody coated with latex microspheres containing fluorescent and dye substances; the R2 reagent is a progesterone peptide modified with a water-soluble biotin derivative; and the R3 reagent is a streptavidin coated with a latex microsphere containing a photosensitive substance; In the R1 reagent, the coating ratio of latex microspheres containing fluorescent and dye substances to progesterone antibodies is 1 mg:5 μg; In the R2 reagent, the labeling ratio of the water-soluble biotin derivative to the progesterone peptide is 5 mol:1 mol; In the R3 reagent, the coating ratio of the latex microspheres containing the photosensitive substance to streptavidin is 1 mg:50 μg; The mass ratio of the progesterone peptide in the R2 reagent to the progesterone antibody in the R1 reagent is 16.67-50:1; The molar ratio of the water-soluble biotin derivative in the R2 reagent to the streptavidin in the R3 reagent is not greater than 5:1 and not less than 4:1; The preparation method of the three-component solid reagent is: R1 reagent, R2 reagent and R3 reagent are diluted with a diluent according to a certain concentration and then freeze-dried; The diluents are H1 buffer and H3 buffer; the components of the H1 buffer are: 50mM-250mM (w / v) TRIS, 0.1%-1.0% (w / v) NaCl, 0.1%-1.0% (w / v) Tween20, 0.1%-1.0% (v / v) Proclin300, 0.1%-5.0% (w / v) bovine serum albumin, pH 6.8-7.4; the components of the H3 buffer are: 100mM-500mM (w / v) citric acid, 100mM-500mM (w / v) sodium citrate, 0.1%-1.0% (w / v) NaCl, 0.1%-1.0% (w / v) Tween20, 0.1%-1.0% (v / v) Proclin300, 0.1%-5.0% (w / v) bovine serum albumin, pH 4.0-5.0; The actual working contents of the progesterone antibody in the R1 reagent, the progesterone peptide in the R2 reagent, and the streptavidin in the R3 reagent after dilution with the diluent are 7 ng / person, 250 ng / person, and 250 ng / person, respectively; The application method of the kit comprises: diluting the sample with a sample diluent, adding the three-component solid reagent in the kit, and after the three-component solid reagent is re-dissolved, the three-component solid reagent and the sample react synchronously, and obtaining the test result after a period of reaction.

2. A method for preparing the kit according to claim 1, characterized in that: The R1 reagent, R2 reagent and R3 reagent are diluted with diluent to a certain concentration and then freeze-dried.

3. The preparation method according to claim 2, characterized in that: The freeze-drying process of the freeze-drying is: pre-freezing at -50°C for 2h, vacuuming at -45°C for 1h, freeze-drying at -40°C for 1h, freeze-drying at -35°C for 8h, freeze-drying at -30°C for 2h, freeze-drying at -20°C for 2h, freeze-drying at -10°C for 2h, freeze-drying at 0°C for 4h, freeze-drying at 10°C for 2h, freeze-drying at 20°C for 2h, freeze-drying at 30°C for 2h and insulation treatment.

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