Chemiluminescence immunoassay method and kit for detecting estrogen of panda

Abstract

The invention discloses a chemiluminescence immunoassay method and kit for detecting estrogen of pandas, and belongs to the technical field of biological detection. The specific process comprises the following steps: by taking goat anti-rabbit IgG as a coating antibody, enabling an estrogen standard substance or a sample to be detected and horse radish peroxidase labeled estrogen to compete with a limited amount of estrogen antibody; after incubation and washing, adding a chemiluminescent substrate, and quantitatively analyzing the estrogen in the urine or excrement of the panda according to a standard curve by detecting the intensity of a luminescent signal. Compared with an existing enzyme-linked immunosorbent assay method, the novel chemiluminescence immunoassay method provided by the invention has higher sensitivity (the lowest detection line can reach 9.75 pg / mL) and shorter detection time (only 40 minutes are needed in the whole process), and a rapid and accurate technical means is provided for oestrus identification and reproduction management of pandas.

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 estrogen in giant pandas. Background Technology

[0002] Captive breeding of giant pandas is a crucial measure for the conservation of this rare species; however, their unique reproductive physiology presents significant challenges to this work. Female giant pandas experience only one estrus cycle per year, and their effective fertility window is extremely short, typically only 24 to 72 hours. This fleeting physiological characteristic means that any misjudgment of timing can lead to the failure of the entire year's breeding efforts. Against this backdrop, precise and rapid monitoring of the core reproductive hormone—estrogen—has become the key technology for overcoming this reproductive bottleneck.

[0003] Estrogen plays a crucial "messenger" role in the estrous cycle of female giant pandas. During proestrus, its level steadily rises, reaching a decisive peak before ovulation. This characteristic hormone surge is the most direct and reliable physiological signal indicating impending follicle maturation and ovulation. Therefore, the fundamental purpose of continuously monitoring the dynamic changes in estrogen levels is to accurately capture this peak, thereby providing the breeding team with a scientific and objective basis for decision-making, identifying the optimal mating or artificial insemination time within the next one to two days. This not only significantly increases the probability of conception in a single mating but also represents a key leap from "experience-based judgment" to "data-driven" refined breeding management.

[0004] Given the extremely short estrus period and rapid hormonal changes in giant pandas, the speed and timeliness of detection technologies are subject to stringent requirements. Traditional methods such as enzyme-linked immunosorbent assay (ELISA), while widely used, are time-consuming, often requiring several hours or even longer. During the critical estrus peak, any delay in test results can lead to delayed decision-making, thus missing the golden window for conception. 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 estrogen 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 estrogen detection that offers higher sensitivity, faster detection speed, and simpler 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 estrogen in giant pandas, comprising the following steps: (1) Preparation of a solid-phase carrier coated with goat anti-rabbit IgG; (2) Add the estrogen standard and the sample to be tested to the solid-phase carrier mentioned above, and then add estrogen antibody and horseradish peroxidase labeled estrogen 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 estrogen standard, and then calculate the estrogen 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-rabbit IgG with coating buffer at a ratio of 1:50~100, 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-rabbit IgG was 1:100.

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

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

[0011] Furthermore, the estrogen antibody was diluted at a ratio of 1:100 to 200, and the horseradish peroxidase-labeled estrogen was diluted at a ratio of 1:300 to 500.

[0012] Furthermore, the working dilution ratio of the estrogen antibody was 1:150.

[0013] Furthermore, the working dilution of horseradish peroxidase-labeled estrogen was 1:400.

[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 20~40 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 estrogen concentration of the sample was calculated based on the emission value of the sample. The concentration gradients of the estrogen standards were: 0, 9.75, 19.5, 39, 78, 156.2, 312.5, 625, 1250, 2500, and 5000 pg / mL.

[0018] Another object of the present invention is to provide a kit for detecting estrogen in giant pandas, comprising a solid-phase carrier pre-coated with goat anti-rabbit IgG, estrogen standard (Sigma, E1752), estrogen antibody / horseradish peroxidase-labeled estrogen (EC522-2), 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 estrogen in giant pandas.

[0020] The beneficial effects of this invention are: 1. This invention, through optimization of the reaction system, achieves a detection limit as low as 9.75 pg / mL, significantly lower than traditional ELISA methods. It can accurately capture minute hormonal changes during proestrus, and the standard curve exhibits a wide linear range (9.75-5000 pg / mL) with a low correlation coefficient R0. 2 >0.99, exhibiting excellent sensitivity.

[0021] 2. The entire detection process constructed by this invention, from sample addition to obtaining results, can be completed within 40 minutes, which significantly shortens the detection time compared to ELISA and can provide timely guidance for breeding practices.

[0022] 3. The reagent kit constructed in this invention has complete components and optimized reaction conditions, making it easy to promote and use in application scenarios such as bases. Attached Figure Description

[0023] Figure 1 Standard curve for chemiluminescent immunoassay of estrogen in giant panda urine; Figure 2 This is the result of a chemiluminescent immunoassay for estrogen 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 estrogen in giant panda urine 1. Coating: Dilute goat anti-rabbit 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 estrogen standards as follows: 0, 9.75, 19.5, 39, 78, 156.2, 312.5, 625, 1250, 2500, and 5000 pg / mL. Add 50 μL of the standard and 1:20 diluted urine sample (the urine dilution concentration depends on the actual situation; in this application, it is diluted 1:20) to the corresponding standard and sample wells, respectively.

[0028] 4. Add antibody and enzyme label: Add 100 μL of estrogen antibody (R522-2) diluted 1:150 and 100 μL of estrogen HRP working solution diluted 1:400 to each well (no antibody is added to blank wells) so that the estrogen standard or test sample (urine) and horseradish peroxidase labeled estrogen compete for a limited amount of estrogen 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 estrogen 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: Enzyme-linked immunosorbent assay (ELISA) for estrogen in giant panda urine 1. Coating: Dilute goat anti-rabbit 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 estrogen standards at 0, 39, 78, 156.2, 312.5, 625, 1250, 2500, and 5000 pg / mL. Add 50 μL of the standard and 1:20 diluted urine sample (the urine dilution concentration depends on the actual situation; in this application, it is diluted 1:20) to the corresponding standard and sample wells, respectively.

[0035] 4. Add antibody and enzyme label: Add 100 μL of estrogen antibody diluted 1:500 and 100 μL of estrogen HRP working solution diluted 1:500 to each well (do not add antibody to blank wells) so that the estrogen standard or test sample (urine) and HRP-labeled estrogen compete for a limited amount of estrogen 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, wash 4 times. Add 100 μL of freshly prepared TMB chromogenic solution 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 luminescence value of each well at a wavelength of 450 nm. Establish a four-parameter logistic regression standard curve using the logarithm of the standard concentration and the luminescence value, calculate the estrogen 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 Estrogen 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 line of this invention can reach 9.75 pg / ml, which is significantly better than the lowest detection line of 39 pg / ml in Comparative Example 1. This indicates that the detection method constructed in this invention can more accurately reflect the changes in giant pandas during estrus. It can be used for detecting estrogen in giant panda reproduction and production, rapidly analyzing the changes in estrus in giant pandas, and assisting in determining the timing of mating.

[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 estrogen in giant pandas, characterized in that, Includes the following steps: (1) Preparation of a solid-phase carrier coated with goat anti-rabbit IgG; (2) Add the estrogen standard and the sample to be tested to the solid-phase carrier mentioned above, and then add estrogen antibody and horseradish peroxidase labeled estrogen 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 estrogen standard, and then calculate the estrogen content in the sample to be tested based on the standard curve.

2. The chemiluminescent immunoassay method for detecting estrogen in giant pandas according to claim 1, characterized in that, The preparation process of step (1) is as follows: Dilute goat anti-rabbit IgG with coating buffer at a ratio of 1:50~100, then add it to the microwells of a chemiluminescent ELISA plate at a rate of 200 μL / well and incubate overnight at 4°C.

3. The chemiluminescent immunoassay method for detecting estrogen in giant pandas according to claim 2, characterized in that, In step (2), estrogen standards and test samples are added to different microwells, and then estrogen antibodies and horseradish peroxidase-labeled estrogen are added simultaneously to conduct a competitive immune response.

4. The chemiluminescent immunoassay method for detecting estrogen in giant pandas according to claim 3, characterized in that, Both the estrogen standard and the test sample were added in an amount of 50 μL.

5. The chemiluminescent immunoassay method for detecting estrogen in giant pandas according to claim 3, characterized in that, Estrogen antibodies were diluted at a ratio of 1:100 to 200, and horseradish peroxidase-labeled estrogens were diluted at a ratio of 1:300 to 500.

6. The chemiluminescent immunoassay method for detecting estrogen in giant pandas according to claim 1, characterized in that, The sample to be tested in step (2) is giant panda excrement.

7. The chemiluminescent immunoassay method for detecting estrogen in giant pandas according to claim 6, characterized in that, The samples to be tested are giant panda urine or feces.

8. The chemiluminescent immunoassay method for detecting estrogen in giant pandas according to claim 1, characterized in that, In step (2), a competitive immune response is carried out at 35~37℃ for 20~40 min.

9. A kit for detecting estrogen in giant pandas, characterized in that, It includes a solid-phase carrier pre-coated with goat anti-rabbit IgG, estrogen standards, estrogen antibody / horseradish peroxidase-labeled estrogen, chemiluminescent substrate solution, and washing solution.

10. Use of the kit according to claim 9 in the detection of estrogen in giant pandas.

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