Simoa triple detection reagents, kits and applications for biomarkers IL-8, hCGβ and RANTES

Abstract

The present invention relates to Simoa triple detection reagents, kits and applications of biomarkers IL-8, hCGβ and RANTES, and to the field of immunoassay technology. The Simoa triple detection reagent includes capture antibody-coated magnetic beads, biotinylated detection antibodies, streptavidin-β-galactosidase solution and fluorescent substrate solution; the magnetic beads coated with the capture antibody of IL-8, the magnetic beads coated with the capture antibody of hCGβ and the magnetic beads coated with the capture antibody of RANTES are magnetic beads with different fluorescence encodings. The Simoa triple detection reagent or kit of the present invention realizes high-throughput detection of multiple protein markers that can evaluate the potential of embryo implantation, realizes simultaneous detection of multiple protein markers in one detection, and provides the possibility for analyzing and studying the relationship between the target protein in the conditioned culture medium of a single blastocyst and the embryo quality and the pregnancy outcome of the transplantation.

Description

Technical Field

[0001] The present invention relates to the field of immunoassay technology, and in particular to a Simoa triple detection reagent, a kit and applications of biomarkers IL-8, hCGβ and RANTES. Background Art

[0002] Human assisted reproductive technology (ART) has developed rapidly and has become the most effective method for treating infertility. Although ART technology is becoming more and more mature, there are still some urgent problems to be solved, such as the high rate of multiple pregnancy. In order to improve the clinical pregnancy rate, live birth rate, and reduce the rate of multiple pregnancy, the selection of high-quality single embryo transplantation has gradually become a trend. At present, embryo morphology scoring is still the most commonly used embryo evaluation method in reproductive centers around the world, but morphology scoring is a subjective judgment method that depends on the experience of embryologists and has poor accuracy. As a supplement to the traditional morphological method for selecting embryos, the Time-lapse morphological dynamics model developed in recent years has improved the pregnancy rate to a certain extent, but the accuracy of embryo selection by this method is still difficult to meet expectations. This shows that the morphological evaluation method still has great limitations. PGT (preimplantation genetic testing) can evaluate whether the embryo is suitable for transplantation at the genetic level, but it is invasive, and its main role in clinical application is eugenics, rather than evaluating the implantation potential of the embryo. Therefore, how to non-invasively select the embryos with the greatest developmental potential for transplantation is still a problem that plagues clinicians and embryologists.

[0003] During in vitro culture, pre-implantation embryos secrete some proteins, whose expression and secretion may be closely related to embryonic development and implantation. Secretory proteins may act on themselves in an autocrine manner to regulate, or they may act on the endometrium in a paracrine manner to achieve maternal-fetal dialogue. Therefore, by detecting embryonic secretory proteins in the culture medium, it is expected to achieve a non-invasive, objective, and accurate assessment of embryonic development potential.

[0004] It is possible to non-invasively predict embryo implantation potential by detecting secretory proteins in embryo conditioned medium. However, the content of embryo secretory proteins is extremely low and the volume is small (25-30 μL), which makes the existing Luminex technology and ELISA technology difficult to detect, and the sensitivity cannot meet the requirements of quantitative detection of single blastocyst conditioned medium and simultaneous detection of multiple secretory proteins in a single sample. In view of this, the present invention provides Simoa triple detection reagents, kits and applications of biomarkers IL-8, hCGβ and RANTES. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a Simoa triple detection reagent, a kit and an application of the biomarkers IL-8, hCGβ and RANTES. The purpose is to develop an ultra-high sensitivity Simoa triple detection kit for IL-8, hCGβ and RANTES to achieve simultaneous and quantitative detection of IL-8, hCGβ and RANTES in a single blastocyst conditioned medium.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] In a first aspect, a Simoa triple detection reagent for biomarkers IL-8, hCGβ and RANTES, the Simoa triple detection reagent comprising capture antibody-coated magnetic beads, biotinylated detection antibodies, streptavidin-β-galactosidase solution (SBG) and a fluorescent substrate solution; the capture antibody-coated magnetic beads comprise IL-8 capture antibody-coated magnetic beads, hCGβ capture antibody-coated magnetic beads and RANTES capture antibody-coated magnetic beads; the magnetic beads among the IL-8 capture antibody-coated magnetic beads, the hCGβ capture antibody-coated magnetic beads and the RANTES capture antibody-coated magnetic beads are magnetic beads with different fluorescent codes; the biotinylated detection antibodies comprise biotinylated IL-8 detection antibodies, biotinylated hCGβ detection antibodies and biotinylated RANTES detection antibodies.

[0008] Based on the above technical solution, the present invention can also be improved as follows.

[0009] Furthermore, the fluorescent substrate solution includes RGP (resorufin-β-D-galactoside).

[0010] Furthermore, the magnetic beads with different fluorescence codes include 488nm magnetic beads, 700nm magnetic beads and 750nm magnetic beads.

[0011] Furthermore, the number ratio of the magnetic beads coated with the IL-8 capture antibody, the magnetic beads coated with the hCGβ capture antibody, and the magnetic beads coated with the RANTES capture antibody is 1:1:1; wherein the total amount of the magnetic beads is 300,000-500,000;

[0012] The concentration of the biotinylated IL-8 detection antibody is 1 μg / mL-3 μg / mL, the concentration of the biotinylated hCGβ detection antibody is 8 μg / mL-10 μg / mL; and the concentration of the biotinylated RANTES detection antibody is 1 μg / mL-3 μg / mL.

[0013] Furthermore, the IL-8 capture antibody-coated magnetic beads are mainly prepared by the following steps: subjecting the IL-8 capture antibody to liquid replacement treatment with a magnetic bead coupling buffer to obtain the IL-8 capture antibody after liquid replacement; activating the first magnetic beads among the different fluorescent encoded magnetic beads to obtain the first activated magnetic beads; coupling the IL-8 capture antibody after liquid replacement with the first activated magnetic beads, washing the coupled product, blocking with a blocking buffer, and then washing with a magnetic bead washing solution to obtain the IL-8 capture antibody-coated magnetic beads;

[0014] Or / and, the hCGβ capture antibody-coated magnetic beads are mainly prepared by the following steps: subjecting the hCGβ capture antibody to liquid replacement treatment with a magnetic bead coupling buffer to obtain the hCGβ capture antibody after liquid replacement; activating the second magnetic beads among the different fluorescent coded magnetic beads to obtain the second activated magnetic beads; coupling the hCGβ capture antibody after liquid replacement with the second activated magnetic beads, washing the coupled product, blocking with a blocking buffer, and then washing with a magnetic bead washing solution to obtain the hCGβ capture antibody-coated magnetic beads;

[0015] Or / and, the magnetic beads coated with the capture antibody of RANTES are mainly prepared by the following steps: subjecting the capture antibody of RANTES to liquid replacement treatment with a magnetic bead coupling buffer to obtain the capture antibody of RANTES after liquid replacement; activating the third magnetic beads among the different fluorescent encoded magnetic beads to obtain the third activated magnetic beads; coupling the capture antibody of RANTES after liquid replacement with the third activated magnetic beads, washing the coupling product, blocking with a blocking buffer, and then washing with a magnetic bead washing solution to obtain the magnetic beads coated with the capture antibody of RANTES.

[0016] Among them, the first magnetic beads, the second magnetic beads and the third magnetic beads of the different fluorescent encoded magnetic beads are any one of 488nm magnetic beads, 700nm magnetic beads and 750nm magnetic beads respectively.

[0017] Furthermore, in the preparation of the IL-8 capture antibody-coated magnetic beads, the concentration of the IL-8 capture antibody in the IL-8 capture antibody after the liquid replacement is adjusted to 0.1 mg / mL-0.3 mg / mL, preferably 0.2 mg / mL, and the concentration of the first activated magnetic beads in the coupling is 1.4×10 9 / mL, the activation reagent used in the first magnetic beads includes EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride), the concentration of EDC is 0.2mg / mL-0.4mg / mL, preferably 0.3mg / mL, the activation temperature is 4°C, the activation time is 20min-40min, preferably 30min; the coupling conditions are: coupling temperature 4°C, coupling time 1h-3h, preferably 2h;

[0018] In the preparation of the hCGβ capture antibody-coated magnetic beads, the concentration of the hCGβ capture antibody in the hCGβ capture antibody after liquid replacement is adjusted to 0.1 mg / mL-0.3 mg / mL, preferably 0.2 mg / mL, and the concentration of the second activated magnetic beads in the coupling is 1.4×10 9 / mL, the activation reagent used in the second magnetic beads includes EDC, the concentration of the EDC is 0.2mg / mL-0.4mg / mL, preferably 0.3mg / mL, the activation temperature is 4°C, the activation time is 20min-40min, preferably 30min; the coupling conditions are: coupling temperature 4°C, coupling time 1h-3h, preferably 2h;

[0019] In the preparation of the RANTES capture antibody-coated magnetic beads, the concentration of the RANTES capture antibody in the RANTES capture antibody after liquid replacement is adjusted to 0.1 mg / mL-0.3 mg / mL, preferably 0.2 mg / mL, and the concentration of the third activated magnetic beads in the coupling is 1.4×10 9 / mL, the activation reagent used in the third magnetic beads includes EDC, the concentration of the EDC is 0.2mg / mL-0.4mg / mL, preferably 0.3mg / mL, the activation temperature is 4°C, the activation time is 20min-40min, preferably 30min; the coupling conditions are: coupling temperature 4°C, coupling time 1h-3h, preferably 2h.

[0020] Furthermore, the preparation method of the biotinylated detection antibody comprises: subjecting the detection antibody to liquid replacement treatment with a biotinylation reaction buffer, the detection antibody comprising an IL-8 detection antibody, an hCGβ detection antibody and a RANTES detection antibody, and then connecting the replaced detection antibody with biotin, and purifying the obtained product to obtain a biotinylated IL-8 detection antibody, a biotinylated hCGβ detection antibody and a biotinylated RANTES detection antibody, respectively.

[0021] Furthermore, the biotin in the biotinylated IL-8 detection antibody, the biotinylated hCGβ detection antibody, and the biotinylated RANTES detection antibody all include NHS-Biotin;

[0022] The molar ratio of the biotinylated IL-8 detection antibody to NHS-Biotin is 1:20-60, preferably 1:40; the molar ratio of the biotinylated hCGβ detection antibody to NHS-Biotin is 1:20-60, preferably 1:40; the molar ratio of the biotinylated RANTES detection antibody to NHS-Biotin is 1:20-60, preferably 1:40.

[0023] Furthermore, the Simoa triple detection reagent also includes an IL-8 standard, an hCGβ standard, a RANTES standard and a sample diluent.

[0024] In a second aspect, a Simoa triple detection kit for biomarkers IL-8, hCGβ and RANTES, wherein the Simoa triple detection kit comprises the Simoa triple detection reagents for the biomarkers IL-8, hCGβ and RANTES.

[0025] In a third aspect, the use of the Simoa triple detection reagent for the biomarkers IL-8, hCGβ and RANTES or the Simoa triple detection kit for the biomarkers IL-8, hCGβ and RANTES in the preparation of a detection reagent for detecting the content of IL-8, hCGβ and RANTES in the conditioned culture medium of a single human embryo.

[0026] The single molecule array technology (Simoa) used in the present invention is a cutting-edge technology in the field of protein detection by immunoassay. Its characteristics are: (1) the capture antibody is coated on the magnetic beads, and a sufficient amount of magnetic beads meet the low concentration of target molecules. According to the Poisson distribution principle, 1 or 0 target molecules are captured on each magnetic bead; (2) each magnetic bead falls into a 4.2μm microwell, and each well can only accommodate one magnetic bead; (3) the single enzyme contained in the magnetic beads of the immune complex can catalyze the substrate molecule (RGP) to produce a large amount of fluorescent substances; (4) the target molecule is quantified by counting the number of luminescent microwells. Therefore, since the fluorescent product is concentrated in the extremely small microwell, the fluorescence imaging of a single microwell is realized in combination with the fluorescence microscope imaging technology, thereby realizing the detection of a single protein molecule, which greatly improves the sensitivity of the detection.

[0027] The beneficial effects of the present invention are:

[0028] (1) The Simoa triple detection reagent or kit of the present invention realizes high-throughput detection of multiple protein markers that can evaluate embryo implantation potential, and realizes simultaneous detection of multiple protein markers in one detection, providing the possibility for analyzing and studying the relationship between the target protein in the conditioned culture medium of a single blastocyst and the embryo quality and the pregnancy outcome of the transplantation.

[0029] (2) The present invention meets the detection requirements of samples with small sample volume and extremely low protein abundance by improving the detection sensitivity of the kit.

[0030] (3) The triple test kit of the present invention is combined with the fully automatic Simoa single molecule immunoassay array analyzer to achieve higher detection accuracy and precision. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a schematic diagram of the detection principle of the IL-8, hCGβ and RANTES Simoa triple detection kit of the present invention;

[0032] Figure 2 This is a standard curve diagram of the Simoa triple detection kit for IL-8 of the present invention;

[0033] Figure 3 The standard curve diagram of the Simoa triple detection kit for hCGβ of the present invention;

[0034] Figure 4 It is a standard curve diagram of the Simoa triple detection kit for RANTES of the present invention. DETAILED DESCRIPTION

[0035] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0036] Sources of materials and reagents:

[0037] Example

[0038] 1. Simoa triple detection reagent and preparation of biomarkers IL-8, hCGβ and RANTES.

[0039] A Simoa triple detection reagent for biomarkers IL-8, hCGβ and RANTES, wherein the Simoa triple detection reagent comprises capture antibody-coated magnetic beads, biotinylated detection antibodies, streptavidin-β-galactosidase solution (SBG), fluorescent substrate solution (RGP, resorufin-β-D-galactoside), IL-8 standard, hCGβ standard, RANTES standard, sample diluent, magnetic bead diluent, and SBG diluent; the capture antibody-coated magnetic beads comprise IL-8 capture antibody-coated magnetic beads, hCGβ capture antibody-coated magnetic beads and RANTES capture antibody-coated magnetic beads; the magnetic beads in the IL-8 capture antibody-coated magnetic beads, the hCGβ capture antibody-coated magnetic beads and the RANTES capture antibody-coated magnetic beads are magnetic beads with different fluorescence codes; the biotinylated detection antibodies comprise biotinylated IL-8 detection antibodies, biotinylated hCGβ detection antibodies, biotinylated RANTES detection antibodies ( Figure 1 ).

[0040] The specific implementation scheme of Simoa triple detection reagent includes the following steps: preparing magnetic bead-coupled capture antibody reagent, biotinylated detection antibody reagent, configuring IL-8, hCGβ and RANTES standard dilution solution, diluting SBG reagent and pre-treatment with single blastocyst conditioned medium.

[0041] (1) Preparation of the magnetic bead-coupled capture antibody:

[0042] The preparation method of the magnetic bead-coupled capture antibody is as follows:

[0043] 1) Using magnetic bead coupling buffer (BCB) to perform liquid replacement treatment on IL-8, hCGβ, and RANTES capture antibodies through ultrafiltration tubes, and collecting the capture antibodies after IL-8, hCGβ, and RANTES replacement of liquid respectively;

[0044] 2) Three fluorescent coded magnetic beads (488nm, 700nm, 750nm) each (1.4×10 9 After repeated washing with magnetic bead washing buffer (BWB), the magnetic beads were resuspended in BCB and mixed with 1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC, 0.3 mg / mL) at 4°C for 30 min to activate the magnetic beads.

[0045] 3) After the activated magnetic beads were washed with BCB, the activated magnetic beads with different fluorescence codes were coupled with different capture antibodies (0.2 mg / mL) at 4°C for 2 h;

[0046] 4) After washing the magnetic beads with BWB for 3 times, the uncoupled sites were blocked with magnetic bead blocking buffer (BBB) ​​at room temperature for 45 minutes; finally, the beads were washed with BWB and stored with magnetic bead diluent to obtain magnetic beads with different fluorescence codes coated with different capture antibodies, namely, magnetic beads coated with IL-8 capture antibody, magnetic beads coated with hCGβ capture antibody and magnetic beads coated with RANTES capture antibody.

[0047] (2) Preparation of the biotinylated IL-8, hCGβ and RANTES detection antibodies:

[0048] The preparation method of the biotinylated IL-8, hCGβ and RANTES detection antibodies is as follows:

[0049] 1) IL-8, hCGβ, and RANTES detection antibodies were replaced with biotinylation reaction buffer (BRB) through ultrafiltration tubes to replace the antibody solvent;

[0050] 2) After replacing the solvent, the detection antibody (1 mg / mL) was mixed with NHS-Biotin and allowed to react at room temperature for 30 minutes;

[0051] 3) After the biotinylation reaction, the biotinylated IL-8 detection antibody, the biotinylated hCGβ detection antibody, and the biotinylated RANTES detection antibody stored in BRB are obtained. The molar ratio of each of the above detection antibodies to NHS-Biotin is 1:40.

[0052] (3) Preparation of dilution solutions of IL-8, hCGβ and RANTES standards:

[0053] The preparation method of the IL-8, hCGβ and RANTES standard dilution solution is as follows:

[0054] 1) Prepare IL-8 standard gradient solution, dilute IL-8 standard of known concentration with sample diluent (120 pg / mL, 40 pg / mL, 13.3 pg / mL, 4.4 pg / mL, 1.48 pg / mL, 0.49 pg / mL, 0.17 pg / mL, 0 pg / mL);

[0055] 2) Prepare a gradient solution of hCGβ standard, dilute the known concentration of hCGβ standard with sample diluent (135mIU / mL, 43.55mIU / mL, 14.05mIU / mL, 4.53mIU / mL, 1.46mIU / mL, 0.47mIU / mL, 0.15mIU / mL, 0mIU / mL);

[0056] 3) Prepare a gradient solution of RANTES standard, and dilute RANTES standard of known concentration with sample diluent (48 pg / mL, 19.2 pg / mL, 7.68 pg / mL, 3.07 pg / mL, 1.23 pg / mL, 0.49 pg / mL, 0.20 pg / mL, 0 pg / mL).

[0057] The concentration points of the three standards were mixed at a ratio of 1:1:1, that is, the final three-factor standard curve was the IL-8 standard curve (40pg / mL, 13.3pg / mL, 4.4pg / mL, 1.48pg / mL, 0.49pg / mL, 0.17pg / mL, 0.055pg / mL, 0pg / mL); the hCGβ standard curve (45mIU / mL, 14.52, 4.68mIU / mL) / mL, 1.51mIU / mL, 0.49mIU / mL, 0.16mIU / mL, 0.051mIU / mL, 0mIU / mL); RANTES standard curve (16pg / mL, 6.4pg / mL, 2.56pg / mL, 1.02pg / mL, 0.41pg / mL, 0.164pg / mL, 0.066pg / mL, 0pg / mL).

[0058] (4) Preparation of the diluted SBG reagent:

[0059] The preparation method of the diluted SBG reagent is as follows: diluting the SBG reagent, diluting the SBG mother solution to 150PM by using the SBG diluent as the working solution.

[0060] (5) Pre-treatment of the single blastocyst conditioned medium sample:

[0061] The method for preparing the single blastocyst conditioned medium sample pretreatment is as follows: measuring the volume of the single blastocyst conditioned medium sample, adding the sample diluent to a total volume of 130ul, recording the data and calculating the dilution multiple.

[0062] Note: The above three fluorescent coded magnetic beads, BCB, BWB, BBB, BRB, NHS-Biotin, EDC, SBG, RGP, sample diluent, magnetic bead diluent, and SBG diluent are all supporting reagents provided by Simoa's homemade kits.

[0063] 2. IL-8, hCGβ and RANTES Simoa triple detection kit and usage.

[0064] (1) Simoa Triple Detection Kit:

[0065] A Simoa triple detection kit for biomarkers IL-8, hCGβ and RANTES comprises the Simoa triple detection reagent for biomarkers IL-8, hCGβ and RANTES.

[0066] (2) Use of the kit:

[0067] The method for using the Simoa triple detection kit for biomarkers IL-8, hCGβ and RANTES includes the following steps:

[0068] 1) First, import the electronic file of the multiple detection test method produced by Quanterix on the Quanterix HD-X instrument; set the experimental conditions: in the first step, mix the capture antibody-coated magnetic beads (25 μL / sample), standard / sample (100 μL / sample), and biotinylated detection antibody (20 μL / sample) and incubate for 35 minutes; in the second step, add SBG working solution (100 μL / sample) and incubate for 5 minutes; set the concentration points of the standard curve of each factor as described above; select the fluorescence channel for each factor detection according to the actual situation of fluorescence-encoded magnetic beads coupled to capture antibodies.

[0069] 2) Then prepare a sufficient amount of mixed solution of magnetic beads coated with different capture antibodies (total magnetic bead concentration 2×10 7 All reagents were loaded into the instrument together: 1 μg / mL, 1 μg / mL biotinylated detection antibody mixed solution (IL-8: 1 μg / mL, hCGβ: 10 μg / mL, RANTES: 1 μg / mL), SBG working solution (150 PM), RGP solution (activated by vibration at 30 °C for 30 min), and a 96-well plate with standards and samples.

[0070] 3) Finally, check whether the consumables and reagents are sufficient. If they are sufficient, run the test and export the fitting curve and sample test results after completion.

[0071] 3. Experiments and results.

[0072] (1) Standard curve, detection limit and detection limit:

[0073] The IL-8, hCGβ and RANTES Simoa triple detection kit developed by the present invention has the standard curve automatically fitted by the instrument, such as Figures 2 to 4 As shown. According to the four-parameter fitting equation of the standard curve: IL-8 standard curve R in triple detection kit 2 is 0.990, where the four-parameter fitting equation of the standard curve is A=0.0195, B=0.998, C=309.5, D=73.3; hCGβ standard curve R 2The R2 of the standard curve of RANTES was 0.999, where A=0.0162, B=0.970, C=552.6, and D=118.6 in the four-parameter fitting equation of the standard curve; the R2 of the standard curve of RANTES was 0.994, where A=0.0453, B=0.918, C=112.3, and D=35.1 in the four-parameter fitting equation of the standard curve. The calculated results showed that the limit of detection (LOD) of IL-8 was 0.024pg / mL, and the limit of detection (LOQ) was 0.056pg / mL; the LOD of hCGβ was 0.017mIU / mL, and the LOQ was 0.043mIU / mL; the LOD of RANTES was 0.022pg / mL, and the LOQ was 0.055pg / mL (as shown in Table 1).

[0074] Table 1 LOD, LOQ and R of the standard curve of each factor 2 value

[0075]

[0076] (2) Accuracy and precision:

[0077] The IL-8, hCGβ and RANTES Simoa triple detection kit developed by the present invention has good accuracy and precision. By mixing standard samples with different factor concentrations to make quality control samples, the quality control samples are also frozen at -80°C and pre-treated in the same way as the test samples to test the accuracy and precision of the kit. A total of 7 quality control samples with different concentrations were designed for the experiment, and at least 3 replicates were tested for each quality control sample. The average detection concentration of the quality control samples of each concentration should be within the range of ±20% of the labeled value, that is, the calculated deviation (Bias, %) represents the inaccuracy. The percentage coefficient of variation (CV%) represents the imprecision of the kit, and CV% should not exceed 20%. The results show (as shown in Table 2) that the absolute value and CV% value of the Bias% of the quality control samples of each concentration are within 20%, indicating that the kit has good accuracy and precision.

[0078] Table 2 Results of accuracy evaluation of IL-8, hCGβ and RANTES Simoa triple detection kit

[0079]

[0080] The IL-8, hCGβ and RANTES Simoa triple detection kit developed by the present invention has good precision in the detection between kits within a batch. By mixing standard products with different factor concentrations to make quality control samples, the quality control samples are also frozen at -80°C and pre-treated in the same way as the test samples to test the precision of the kit within a batch. The experiment is designed to use 3 kits from the same batch to detect quality control samples. The quality control samples are divided into 3 concentrations of low, medium and high, and each quality control sample is tested in at least 3 replicates. The coefficient of variation (CV%) represents the imprecision of the kit, and the CV% should not exceed 20%. The results show (as shown in Table 3) that the CV% values ​​of the quality control samples of each concentration are all within 20%, indicating that the kit has good precision in the detection between kits within a batch.

[0081] Table 3 Intra-batch precision evaluation results of IL-8, hCGβ and RANTES Simoa triple detection kit

[0082]

[0083] The IL-8, hCGβ and RANTES Simoa triple detection kit developed by the present invention has good precision in the detection between different batches of kits. By mixing standard products with different factor concentrations to make quality control samples, the quality control samples are also frozen at -80°C and pre-treated in the same way as the test samples to test the precision of the kit within the batch. The experiment was designed to use 2 different batches of kits to detect quality control samples. The quality control samples are divided into 3 concentrations of low, medium and high, and each quality control sample is tested in at least 3 replicates. The coefficient of variation (CV%) represents the imprecision of the kit, and the CV% should not exceed 20%. The results show (as shown in Table 4) that the CV% values ​​of the quality control samples of each concentration are all within 20%, indicating that the kit of the invention has good precision in the detection of the kit between batches.

[0084] Table 4 Results of inter-batch precision evaluation of IL-8, hCGβ and RANTES Simoa triple detection kits

[0085]

[0086] (3) Repeatability:

[0087] The IL-8, hCGβ and RANTES Simoa triple detection kit developed by the present invention has good repeatability. Because the volume of the single blastocyst conditioned culture medium is small, only 20-25 μL, in order to verify the repeatability of the test results of the kit for the same sample, multiple single blastocyst conditioned culture mediums are mixed and multiple replicate wells are set to evaluate the repeatability of the IL-8, hCGβ and RANTES Simoa triple detection kit. The above-mentioned single blastocyst conditioned culture medium sample comes from the remaining discarded blastocyst culture medium collected after the in vitro culture of a single blastocyst of an infertile patient undergoing assisted reproductive IVF / ICSI technology. The results show (as shown in Table 5) that the coefficients of variation of IL-8, hCGβ, and RANTES are 9.5%, 10.3%, and 8.8%, respectively. It is proved that the triple detection kit of the present invention has good repeatability.

[0088] Table 5 Repeatability results of testing the same sample (mixed single blastocyst conditioned medium)

[0089]

[0090] (4) Sensitivity:

[0091] The sensitivity of each factor of the IL-8, hCGβ and RANTES Simoa triple detection kit developed by the present invention is improved, and the content of IL-8, hCGβ and RANTES in the single blastocyst conditioned culture medium can be detected simultaneously. A total of 148 single blastocyst conditioned culture medium samples were detected, and the samples were collected from the remaining discarded single blastocyst culture medium after the in vitro culture of embryos of different infertile patients undergoing assisted reproductive IVF / ICSI technology. The results showed that the concentration of RANTES detected in all samples was higher than LOQ; the proportion of samples with IL-8 concentration higher than LOD was 100% (148 / 148), and the proportion of samples higher than LOQ was 83.8% (124 / 148); the proportion of samples with hCGβ concentration higher than LOD was 99.3% (147 / 148), and the proportion of samples higher than LOQ was 68.2% (101 / 148). The sensitivity almost meets the needs of quantitative detection of ultra-low concentrations of IL-8, hCGβ, and RANTES in single blastocyst conditioned culture medium (as shown in Table 6).

[0092] Table 6 Detection results of IL-8, hCGβ and RANTES Simoa triple detection kit for single blastocyst conditioned medium

[0093]

[0094] The IL-8, hCGβ and RANTES Simoa triple detection kit of the present invention adopts a fully automatic detection instrument to solve a series of problems existing in manual operation, so that the detection results have good accuracy and precision, and effectively improve the sensitivity of detection, providing an effective quantitative detection method for samples with small sample volume and extremely low IL-8, hCGβ and RANTES protein content.

[0095] In summary, the Simoa triple detection reagent or kit of the present invention realizes high-throughput detection of multiple protein markers that can evaluate embryo implantation potential, and realizes simultaneous and quantitative detection of multiple protein markers in one detection, providing the possibility for analyzing and studying the relationship between the target protein in the single blastocyst conditioned medium and the embryo quality and the pregnancy outcome of the transplantation. The present invention meets the detection requirements of samples with small sample volume and extremely low protein abundance by improving the detection sensitivity of the kit. The triple kit in the present invention is combined with the fully automatic Simoa single molecule immunoassay array analyzer to obtain higher detection accuracy and precision.

[0096] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. Simoa triple detection reagent for biomarkers IL-8, hCGβ and RANTES, characterized by: The Simoa triple detection reagent includes capture antibody-coated magnetic beads, biotinylated detection antibodies, streptavidin-β-galactosidase solution, and fluorescent substrate solution; the capture antibody-coated magnetic beads include magnetic beads coated with IL-8 capture antibodies, magnetic beads coated with hCGβ capture antibodies, and magnetic beads coated with RANTES capture antibodies; the magnetic beads coated with IL-8 capture antibodies, the magnetic beads coated with hCGβ capture antibodies, and the magnetic beads coated with RANTES capture antibodies are magnetic beads with different fluorescent codes; the biotinylated detection antibodies include biotinylated IL-8 detection antibodies, biotinylated hCGβ detection antibodies, and biotinylated RANTES detection antibodies.

2. The Simoa triple detection reagent for the biomarkers IL-8, hCGβ, and RANTES according to claim 1, characterized in that: The magnetic beads with different fluorescence codes include 488nm magnetic beads, 700nm magnetic beads and 750nm magnetic beads.

3. The Simoa triple detection reagent for the biomarkers IL-8, hCGβ, and RANTES according to claim 1, characterized in that: The number ratio of the magnetic beads coated with the IL-8 capture antibody, the magnetic beads coated with the hCGβ capture antibody, and the magnetic beads coated with the RANTES capture antibody is 1:1:1; The concentration of the biotinylated IL-8 detection antibody is 1 μg / mL-3 μg / mL, the concentration of the biotinylated hCGβ detection antibody is 8 μg / mL-10 μg / mL; and the concentration of the biotinylated RANTES detection antibody is 1 μg / mL-3 μg / mL.

4. The Simoa triple detection reagent for the biomarkers IL-8, hCGβ, and RANTES according to claim 1, characterized in that: The IL-8 capture antibody-coated magnetic beads are mainly prepared by the following steps: subjecting the IL-8 capture antibody to liquid replacement treatment with a magnetic bead coupling buffer to obtain the liquid-replaced IL-8 capture antibody; activating the first type of magnetic beads among the different fluorescent-encoded magnetic beads to obtain the first activated magnetic beads; coupling the liquid-replaced IL-8 capture antibody to the first activated magnetic beads, washing the coupled product, blocking with a blocking buffer, and then washing with a magnetic bead washing solution to obtain the IL-8 capture antibody-coated magnetic beads; Or / and, the hCGβ capture antibody-coated magnetic beads are mainly prepared by the following steps: subjecting the hCGβ capture antibody to liquid replacement treatment with a magnetic bead coupling buffer to obtain liquid-replaced hCGβ capture antibody; activating a second type of magnetic beads among the different fluorescent-encoded magnetic beads to obtain second activated magnetic beads; coupling the liquid-replaced hCGβ capture antibody to the second activated magnetic beads, washing the coupled product, blocking with a blocking buffer, and then washing with a magnetic bead washing solution to obtain hCGβ capture antibody-coated magnetic beads; Or / and, the magnetic beads coated with the RANTES capture antibody are mainly prepared by the following steps: subjecting the RANTES capture antibody to liquid replacement treatment with a magnetic bead coupling buffer to obtain the liquid-replaced RANTES capture antibody; activating the third type of magnetic beads among the different fluorescent-encoded magnetic beads to obtain third activated magnetic beads; coupling the liquid-replaced RANTES capture antibody with the third activated magnetic beads, washing the coupled product, blocking with a blocking buffer, and then washing with a magnetic bead washing solution to obtain the RANTES capture antibody-coated magnetic beads.

5. The Simoa triple detection reagent for the biomarkers IL-8, hCGβ, and RANTES according to claim 4, characterized in that: In the preparation of the IL-8 capture antibody-coated magnetic beads, the concentration of the IL-8 capture antibody in the IL-8 capture antibody after the liquid replacement is adjusted to 0.1 mg / mL-0.3 mg / mL, and the concentration of the first activated magnetic beads in the coupling is 1.4×10 9 / mL, the activation reagent used for the first magnetic beads includes EDC, the concentration of the EDC is 0.2mg / mL-0.4mg / mL, the activation temperature is 4°C, and the activation time is 20min-40min; the coupling conditions are: coupling temperature 4°C, coupling time 1h-3h; In the preparation of the hCGβ capture antibody-coated magnetic beads, the concentration of the hCGβ capture antibody in the hCGβ capture antibody after liquid replacement is adjusted to 0.1 mg / mL-0.3 mg / mL, and the concentration of the second activated magnetic beads in the coupling is 1.4×10 9 / mL, the activation reagent used for the second magnetic beads includes EDC, the concentration of the EDC is 0.2mg / mL-0.4mg / mL, the activation temperature is 4°C, and the activation time is 20min-40min; the coupling conditions are: coupling temperature 4°C, coupling time 1h-3h; In the preparation of the RANTES capture antibody-coated magnetic beads, the concentration of the RANTES capture antibody in the RANTES capture antibody after liquid replacement is adjusted to 0.1 mg / mL-0.3 mg / mL, and the concentration of the third activated magnetic beads in the coupling is 1.4×10 9 / mL, the activation reagent used for the third magnetic beads includes EDC, the concentration of the EDC is 0.2mg / mL-0.4mg / mL, the activation temperature is 4°C, and the activation time is 20min-40min; the coupling conditions are: coupling temperature 4°C, coupling time 1h-3h.

6. The Simoa triple detection reagent for the biomarkers IL-8, hCGβ, and RANTES according to claim 1, characterized in that: The preparation method of the biotinylated detection antibody comprises: performing a liquid replacement treatment on the detection antibody with a biotinylation reaction buffer, wherein the detection antibody includes an IL-8 detection antibody, an hCGβ detection antibody, and a RANTES detection antibody; then linking the replaced detection antibody with biotin; and purifying the obtained product to obtain a biotinylated IL-8 detection antibody, a biotinylated hCGβ detection antibody, and a biotinylated RANTES detection antibody, respectively.

7. The Simoa triple detection reagent for the biomarkers IL-8, hCGβ, and RANTES according to claim 6, characterized in that: The biotin in the biotinylated IL-8 detection antibody, the biotinylated hCGβ detection antibody, and the biotinylated RANTES detection antibody all includes NHS-Biotin; The molar ratio of the biotinylated IL-8 detection antibody to NHS-Biotin is 1:20-60; the molar ratio of the biotinylated hCGβ detection antibody to NHS-Biotin is 1:20-60; and the molar ratio of the biotinylated RANTES detection antibody to NHS-Biotin is 1:20-60.

8. The Simoa triple detection reagent for the biomarkers IL-8, hCGβ, and RANTES according to claim 1, characterized in that: The Simoa triple detection reagent also includes an IL-8 standard, an hCGβ standard, a RANTES standard, and a sample diluent.

9. A Simoa triple detection kit for the biomarkers IL-8, hCGβ, and RANTES, characterized by: The Simoa triple detection kit comprises the Simoa triple detection reagents for the biomarkers IL-8, hCGβ, and RANTES according to any one of claims 1 to 8.

10. Use of the Simoa triple detection reagent for the biomarkers IL-8, hCGβ, and RANTES according to any one of claims 1 to 8 or the Simoa triple detection kit for the biomarkers IL-8, hCGβ, and RANTES according to claim 9 in the preparation of a detection reagent for detecting the levels of IL-8, hCGβ, and RANTES in conditioned medium of a single human embryo.

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