Chemiluminescence immunoassay method and kit for detecting progestational hormone of panda

Abstract

The invention discloses a chemiluminescence immunoassay method and kit for detecting progestational hormone of pandas, and belongs to the technical field of biological detection. The specific process comprises the following steps: by taking goat anti-mouse IgG as a coating antibody, enabling a progestational hormone standard substance or a to-be-detected sample and horse radish peroxidase labeled progestational hormone to compete with a limited amount of progestational hormone antibodies together; after incubation and washing, a chemiluminescent substrate is added, and quantitative analysis of the progestational hormone in the panda urine or excrement is realized by detecting the intensity of a luminescent signal according to a standard curve. The invention provides a novel chemiluminescence immunoassay method, the sensitivity of the chemiluminescence immunoassay method is far higher than that of a traditional ELISA method (the lowest detection line can reach 31.25 pg / mL), the operation is simple and convenient, the detection is rapid, and the urgent demand on rapid and accurate determination of the hormone level in panda pregnancy monitoring and reproduction management can be met.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a chemiluminescent immunoassay method and kit for detecting progesterone in giant pandas. Background Technology

[0002] In the scientific conservation and breeding system of giant pandas, precise monitoring of reproductive endocrine function forms the cornerstone of successful reproduction. Tracking estrogen levels is like observing the smoke from a starting gun; its peak indicates that ovulation may be imminent. The subsequent changes in progesterone levels are the authoritative signal confirming that ovulation has indeed occurred. Continuous monitoring of these two hormones together forms a complete closed loop from prediction to confirmation, providing an indispensable double guarantee for efficient reproductive management.

[0003] In reproductive management practice, the core role of progesterone is primarily in the authoritative confirmation of successful ovulation. While the estrus behavior and estrogen surge in female giant pandas are strong signals guiding mating, confirming the completion of the crucial physiological event of ovulation requires more conclusive evidence. After ovulation, the ruptured follicle transforms into a functional corpus luteum and begins to secrete large amounts of progesterone. Therefore, within approximately 24 hours after ovulation, a significant and sustained rise in progesterone levels from baseline can physiologically confirm successful ovulation and normal corpus luteum function. This crucial endocrine evidence not only validates the accuracy of previous estrogen-based estrus predictions, completing the first key confirmation of the reproductive cycle, but also lays a solid physiological foundation for subsequent pregnancy diagnosis.

[0004] Once ovulation and normal luteal function are confirmed, the core task of progesterone monitoring naturally transitions to pregnancy diagnosis and progress monitoring. In a true pregnancy, the mother must maintain a high and stable progesterone environment to support embryo implantation and fetal development. Therefore, a consistently high progesterone curve generated through regular monitoring becomes the most reliable physiological indicator for assessing pregnancy maintenance and predicting the development of a new life. Summary of the Invention

[0005] To address the aforementioned shortcomings in the existing technology, this invention provides a chemiluminescent immunoassay method and kit for detecting progesterone in giant pandas. The aim is to overcome the deficiencies of existing ELISA technology and provide a chemiluminescent immunoassay method and kit specifically designed for giant panda progesterone, offering higher sensitivity, faster detection speed, and easier operation.

[0006] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows: The purpose of this invention is to provide a chemiluminescent immunoassay method for detecting progesterone in giant pandas, comprising the following steps: (1) Preparation of a solid-phase carrier coated with goat anti-mouse IgG; (2) Add the progesterone standard and the sample to be tested to the solid-phase carrier mentioned above, then add progesterone antibody and horseradish peroxidase labeled progesterone to carry out competitive immune reaction. After the reaction is completed, wash them separately, then add chemiluminescent substrate and detect its luminescence signal. (3) Construct a standard curve based on the luminescence signal detection results of the progesterone standard, and then calculate the progesterone content in the sample to be tested based on the standard curve.

[0007] Furthermore, the preparation process of step (1) is as follows: Dilute goat anti-mouse IgG with coating buffer at a ratio of 1:500~2000, then add it to the microwells of a chemiluminescent ELISA plate at a rate of 200 μL / well and incubate overnight at 4°C.

[0008] Furthermore, the coating dilution ratio of goat anti-mouse IgG was 1:1000.

[0009] Further, in step (2), progesterone standards and test samples are added to different microwells, and then progesterone antibodies and horseradish peroxidase-labeled progesterone are added simultaneously to conduct a competitive immune response.

[0010] Furthermore, the amount of progestin standard and the sample to be tested added was 50 μL.

[0011] Furthermore, the progesterone antibody was diluted at a ratio of 1:200-300, and the horseradish peroxidase-labeled progesterone was diluted at a ratio of 1:250-350.

[0012] Furthermore, the working dilution ratio of the progesterone antibody was 1:240.

[0013] Furthermore, the working dilution of horseradish peroxidase-labeled progesterone was 1:300.

[0014] Furthermore, the sample to be tested in step (2) is giant panda excrement.

[0015] Furthermore, the samples to be tested were giant panda urine or feces.

[0016] Furthermore, in step (2), a competitive immune response is carried out at 35~37℃ for 25~35 min.

[0017] Furthermore, the specific process of step (3) is as follows: The emission value of each well at 425 nm was measured on a chemiluminescence analyzer; a standard curve was plotted with the logarithm of the standard concentration as the x-axis and the corresponding emission value as the y-axis, and the progestin concentration of the sample was calculated based on the emission value of the sample. The concentration gradients of the progestin standards are: 0, 31.25, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000, and 16000 pg / mL.

[0018] Another object of the present invention is to provide a kit for detecting progesterone in giant pandas, comprising a solid-phase carrier pre-coated with goat anti-mouse IgG, progesterone standard (Sigma, P6288), progesterone antibody, horseradish peroxidase-labeled progesterone (CL425), chemiluminescent substrate solution (Roche, 11582950001), and washing solution (phosphate buffer plus Tween 20).

[0019] Another object of the present invention is to provide the use of the above-described kit in the detection of progesterone in giant pandas.

[0020] The beneficial effects of this invention are: 1. By optimizing the reaction system, this invention significantly lowers the detection limit of progesterone, enabling more accurate reflection of subtle changes in hormone levels during early pregnancy and throughout the pregnancy.

[0021] 2. The entire detection process constructed by this invention, from sample addition to obtaining results, can be completed within 35 minutes, which significantly shortens the detection time compared to ELISA, and is conducive to the rapid issuance of test reports to guide breeding management.

[0022] 3. The method of this invention has high repeatability, small inter-batch and intra-batch differences, reliable results, and is easy to promote and apply in non-laboratory environments. Attached Figure Description

[0023] Figure 1 Standard curve for chemiluminescent immunoassay of progesterone in giant panda urine; Figure 2 This is the result of chemiluminescent immunoassay for progesterone detection in the urine of a giant panda. Detailed Implementation

[0024] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0025] Example 1: Chemiluminescence detection of progesterone in giant panda urine 1. Coating: Dilute goat anti-mouse IgG 1:100 with Coating Buffer, add 200 μL to each well, and incubate overnight at 4°C.

[0026] 2. Washing: Discard the liquid inside the hole and wash 3 times with Wash Solution.

[0027] 3. Adding Standards and Samples: Set up a concentration gradient of progesterone standards as follows: 0, 31.25, 62.5, 125, 250, 500, 1000, 2000, 4000, 8000, and 16000 pg / mL. Add 50 μL of the standard and 1:20 diluted urine sample (the urine dilution concentration depends on the actual situation; this application uses a 1:20 dilution ratio) to the corresponding standard and sample wells, respectively.

[0028] 4. Add antibody and enzyme label: Add 100 μL of progestin HRP diluted 1:300 and 100 μL of progestin antibody (CL425) diluted 1:240 to each well (no antibody is added to blank wells) so that the progestin standard or test sample (urine) and horseradish peroxidase-labeled progestin compete for a limited amount of progestin antibody.

[0029] 5. Incubation: Cover with a sealing film and incubate at 37°C in the dark for 30 minutes.

[0030] 6. Washing and luminescence: After the reaction is complete, wash 4 times. Add 100 μL of chemiluminescent substrate solution (Roche, 11582950001) to each well and react at room temperature in the dark for 5 minutes.

[0031] 7. Reading and Calculation: Immediately use a chemiluminescence analyzer to detect the luminescence value of each well at a wavelength of 425 nm. Establish a four-parameter logistic regression standard curve using the logarithm of the standard concentration and the luminescence value. Calculate the progesterone concentration in the sample and correct the final concentration using the sample creatinine. The results are shown in [Figure Number]. Figure 1 , Figure 2 See Table 1.

[0032] Comparative Example 1: Progesterone levels in giant panda urine were determined using routine enzyme-linked immunosorbent assay (ELISA). 1. Coating: Dilute goat anti-mouse IgG 1:100 with Coating Buffer, add 200 μL to each well, and incubate overnight at 4°C.

[0033] 2. Washing: Discard the liquid inside the hole and wash 3 times with Wash Solution.

[0034] 3. Adding Standards and Samples: Set up a concentration gradient of progestin standards at 0, 125, 250, 500, 1000, 2000, 4000, 8000, and 16000 pg / mL. Add 50 μL of the standard and 1:20 diluted urine sample (the urine dilution concentration depends on the actual situation; this application uses a 1:20 dilution ratio) to the corresponding standard and sample wells, respectively.

[0035] 4. Add antibody and enzyme label: Add 100 μL of progestin HRP diluted 1:500 and 100 μL of progestin antibody (CL425) diluted 1:500 to each well (no antibody is added to blank wells) so that the progestin standard or test sample (urine) and horseradish peroxidase labeled progestin compete for a limited amount of progestin antibody.

[0036] 5. Incubation: Cover with a sealing film and incubate at 37°C in the dark for 30 minutes.

[0037] 6. Washing and luminescence: After the reaction is complete, wash 4 times. Add 100 μL of freshly prepared chemiluminescent substrate solution (TMB) to each well and react at room temperature in the dark for 15 minutes.

[0038] 7. Reading and Calculation: Immediately use a chemiluminescence analyzer to detect the absorbance value of each well at a wavelength of 425 nm. Establish a four-parameter logistic curve fitting the standard curve using the logarithm of the standard concentration and the percentage of absorbance to the blank. Calculate the progesterone concentration in the sample and correct the final concentration with the sample creatinine. The results are shown in Table 1.

[0039] Table 1 Sensitivity of Progesterone Detection

[0040] like Figure 1 , Figure 2 As shown in Table 1, the correlation coefficient R of the standard curve of the detection method constructed in this invention is... 2 The value is >0.99, with the horizontal axis representing the logarithm of the standard curve concentration (base 10) and the vertical axis representing the luminescence value and blank percentage. The lowest detection limit of this invention can reach 31.25 pg / mL, which is significantly better than the lowest detection limit of 125 pg / mL of the conventional enzyme-linked immunosorbent assay used in Comparative Example 1. This indicates that the method of this invention can more accurately reflect the changes in giant pandas during estrus and pregnancy, and can be used for the detection of progesterone in giant panda reproduction and production.

[0041] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A chemiluminescent immunoassay method for detecting pregnancy hormone of giant panda, characterized in that, The method comprises the following steps: (1) preparing a solid carrier coated with goat anti-mouse IgG; (2) adding a progesterone standard and a sample to be tested into the solid carrier, then adding a progesterone antibody and a horseradish peroxidase-labeled progesterone to perform a competitive immune reaction, after the reaction, washing is performed, then a chemiluminescent substrate is added and the luminescent signal is detected; (3) constructing a standard curve according to the detection result of the luminescent signal of the progesterone standard, and then calculating the progesterone content in the sample to be tested based on the standard curve.

2. The chemiluminescent immunoassay method for detecting pregnancy hormone of Ailuropoda melanoleuca according to claim 1, characterized in that, The preparation process of step (1) is as follows: The goat anti-mouse IgG is diluted at a ratio of 1:500-2000 with a coating buffer, and then 200 μL / well is added to the micro-wells of the chemiluminescence enzyme-labeled plate, and incubated at 4°C overnight.

3. The chemiluminescent immunoassay method for detecting pregnancy hormone of A. melanoleuca according to claim 2, characterized in that, In step (2), the progesterone standard and the sample to be tested are added to different micro-wells, and then the progesterone antibody and the horseradish peroxidase-labeled progesterone are added to perform a competitive immune reaction.

4. The chemiluminescent immunoassay method for detecting pregnancy hormone of A. melanoleuca according to claim 3, characterized in that, The progesterone standard and the sample to be tested are added in an amount of 50 μL.

5. The chemiluminescent immunoassay method for detecting pregnancy hormone of A. melanoleuca according to claim 3, characterized in that, The progesterone antibody is diluted at a ratio of 1:200-300, and the horseradish peroxidase-labeled progesterone is diluted at a ratio of 1:250-350.

6. The chemiluminescent immunoassay method for detecting pregnancy hormone of A. melanoleuca according to claim 1, wherein, In step (2), the sample to be tested is panda excrement.

7. The chemiluminescent immunoassay method for detecting pregnancy hormone of A. melanoleuca according to claim 6, characterized in that, The sample to be tested is panda urine or feces.

8. The chemiluminescent immunoassay method for detecting pregnancy hormone of A. melanoleuca according to claim 1, wherein, In step (2), the competitive immune reaction is performed at 35-37°C, and the reaction time is 25-35 min.

9. A kit for detecting pregnancy hormone of Ailuropoda melanoleuca, characterized in that, The kit comprises a solid carrier pre-coated with goat anti-mouse IgG, a progesterone standard, a progesterone antibody, a horseradish peroxidase-labeled progesterone, a chemiluminescent substrate solution, and a washing solution.

10. Use of the kit of claim 9 in panda progesterone detection.

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