Polypeptide, kit and method for detecting mouse statin

Abstract

The invention relates to the technical field of biological detection, in particular to polypeptide, a kit and a method for detecting mouse statin. The invention provides a specific synthetic polypeptide with an amino acid sequence as shown in SEQ ID NO: 1, and the synthetic polypeptide is used as a substance with clear chemical definition and replaces natural protein which is complex in structure and difficult to preserve in a traditional double-antibody sandwich method, so that inherent inter-batch difference and quality fluctuation of a biological source standard substance are eliminated from the source. Based on the core reagent, the competitive ELISA detection system constructed by the invention achieves the technical effects of strong specificity, high accuracy and good repeatability, and meanwhile, as the synthetic polypeptide can realize low-cost and high-throughput chemical synthesis, the development period of the whole detection scheme is greatly shortened, and the economical efficiency is remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, specifically to a polypeptide, kit, and method for detecting mouse stomatin. Background Technology

[0002] Inhibin is a heterodimeric glycoprotein hormone primarily secreted by the gonads. Its physiological function is to selectively inhibit the synthesis and secretion of pituitary follicle-stimulating hormone (FSH), playing a central role in reproductive regulation in mice. Precise quantification of inhibin levels in the blood is crucial for basic research in reproductive biology, the evaluation of models of reproductive endocrine diseases, and the development of related drugs.

[0003] Currently, the mainstream technique for quantifying inhibin in mouse blood is enzyme-linked immunosorbent assay (ELISA), with the double-antibody sandwich method being the most common. This method relies on two high-affinity, high-specificity antibodies that can simultaneously recognize different epitopes on the inhibin molecule. One antibody is used to coat a solid-phase carrier to capture the target molecule, while the other enzyme-labeled antibody is used for detection and signal amplification. Although this technique has the advantage of high sensitivity, the screening and pairing of antibody pairs is extremely difficult, time-consuming, and costly. Furthermore, batch-to-batch differences between the two antibodies directly affect the stability and reproducibility of the detection. Additionally, this method typically requires the use of structurally complex natural or recombinant inhibin heterodimer proteins as standards. These proteins are difficult to prepare, have poor stability, and are not easily quantified accurately, leading to fluctuations in the standard curve between different batches of experiments, directly affecting the accuracy and comparability of the quantitative results.

[0004] Besides ELISA, Western blotting is occasionally used for the detection of statin. However, this technique is extremely cumbersome and time-consuming, and is essentially a semi-quantitative method with low sensitivity and low throughput, making it completely unsuitable for the rapid and accurate quantification of large numbers of preclinical samples. Summary of the Invention

[0005] The technical problem this invention aims to solve is to overcome the difficulties and high costs associated with the development of existing double-antibody sandwich ELISA methods for detecting mouse statin, which require pairing two highly specific antibodies, and the inaccurate quantification and poor stability resulting from using heterodimeric proteins as standards. This invention provides a peptide, kit, and method for detecting mouse statin.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of the present invention provides a polypeptide for detecting mouse stomatin, the amino acid sequence of which is shown in SEQ ID NO:1.

[0008] This invention provides a specific synthetic polypeptide with the amino acid sequence shown in SEQ ID NO:1. This synthetic polypeptide, as a chemically defined substance, replaces the complex and difficult-to-preserve natural proteins used in traditional double-antibody sandwich assays, thereby eliminating the inherent batch-to-batch variability and quality fluctuations of biological standards at the source. Based on this core reagent, the competitive ELISA detection system constructed in this invention achieves high specificity, high accuracy, and good reproducibility. Furthermore, because the synthetic polypeptide can be chemically synthesized at low cost and high throughput, the development cycle of the entire detection scheme is significantly shortened, and its economic efficiency is significantly improved.

[0009] A second aspect of the present invention provides a kit for quantitative detection of mouse statin, the kit comprising:

[0010] Includes a solid-phase support for the polypeptide of claim 1;

[0011] A series of concentration standards formulated from the polypeptide of claim 1;

[0012] An antibody capable of specifically binding to the polypeptide of claim 1.

[0013] This invention provides a detection kit comprising a solid-phase carrier coated with a specific synthetic polypeptide, a series of concentration standards formulated with the polypeptide, and an antibody. The kit achieves the technical effects of stable raw material sources, controllable quality, and simple operation, which not only significantly reduces the development threshold and production cost, but also ensures high accuracy and excellent repeatability of the detection results.

[0014] Furthermore, the kit also contains enzyme-labeled streptavidin.

[0015] Furthermore, the enzyme is horseradish peroxidase.

[0016] Furthermore, the kit also includes a colorimetric solution and a stop solution.

[0017] A third aspect of the present invention provides a method for quantitatively detecting inhibin in mouse blood samples, employing a competitive ELISA method, the method comprising the following steps:

[0018] a. Providing a solid-phase support for coating the polypeptide of claim 1;

[0019] b. Add the series of concentration standard solutions and the pretreated mouse blood samples to be tested into different wells of the solid support, respectively;

[0020] c. Add an antibody against the polypeptide of claim 1, incubate to allow the inhibin in the sample to competitively bind to the antibody against the solid-coated polypeptide; discard the liquid, spin dry, add biotin-labeled antibody, and incubate.

[0021] d. After washing, add enzyme-labeled streptavidin and incubate;

[0022] e. After washing, add the color developing solution to carry out the color development reaction, and then add the stop solution to terminate the reaction;

[0023] f. Detect the absorbance value, and calculate the concentration of inhibin in the sample to be tested based on the standard curve plotted using the series of concentration standard solutions.

[0024] This invention provides a competitive ELISA detection method based on the specific synthetic peptide. The method achieves rapid and accurate quantification of mouse blood samples within one hour by using the core step of competitive binding of the inhibin in the sample to the solid-phase coated peptide with the peptide-specific antibody, combined with the subsequent signal amplification and color development system. It achieves the technical effects of simple operation, fast detection speed, high specificity and good repeatability.

[0025] Furthermore, the colorimetric solution is a TMB colorimetric solution.

[0026] Furthermore, the enzyme-labeled streptavidin is horseradish peroxidase-labeled streptavidin.

[0027] Furthermore, the standard curve is plotted using a double logarithmic four-parameter logic curve fitting method.

[0028] Furthermore, the calculated inhibin concentration is expressed in molar concentration units.

[0029] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0030] 1. This invention provides a specific synthetic polypeptide with an amino acid sequence as shown in SEQ ID NO:1. This synthetic polypeptide, as a chemically defined substance, replaces the complex and difficult-to-preserve natural proteins in the traditional double-antibody sandwich method, thereby eliminating the inherent batch-to-batch variability and quality fluctuations of biological standards at the source. Based on this core reagent, the competitive ELISA detection system constructed by this invention achieves high specificity, high accuracy, and good reproducibility. Furthermore, because the synthetic polypeptide can be chemically synthesized at low cost and high throughput, the development cycle of the entire detection scheme is significantly shortened, and its economic efficiency is significantly improved.

[0031] 2. This invention provides a detection kit comprising a solid-phase carrier coated with a specific synthetic polypeptide, a series of concentration standards formulated with the polypeptide, and an antibody. The kit achieves the technical effects of stable raw material sources, controllable quality, and simple operation, which not only significantly reduces the development threshold and production cost, but also ensures high accuracy and excellent repeatability of the detection results.

[0032] 3. This invention provides a competitive ELISA detection method based on the specific synthetic polypeptide. This method achieves rapid and accurate quantification of mouse blood samples within one hour by using the core step of competitive binding of the inhibin in the sample to the solid-phase coated polypeptide with the polypeptide-specific antibody, combined with the subsequent signal amplification and color development system. It achieves the technical effects of simple operation, fast detection speed, high specificity and good repeatability. Attached Figure Description

[0033] Figure 1 This is a diagram showing the predicted linear epitopes of the antigenicity of the inhibin-alpha subunit.

[0034] Figure 2 Comparative images of the effects of superovulation experiments in mice using antibodies with different sequences (embryo morphology).

[0035] Figure 3 A statistical analysis of mouse superovulation experiments using antibodies with different sequences. Detailed Implementation

[0036] The present invention will now be described in detail with reference to the accompanying drawings.

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0038] The first aspect of this embodiment provides a polypeptide for detecting mouse stomatin, the amino acid sequence of which is shown in SEQ ID NO:1.

[0039] The amino acid sequence is as shown in SEQ ID NO:1:

[0040] FRPSQHTRSRQVTSA.

[0041] This embodiment provides a specific synthetic polypeptide with the amino acid sequence shown in SEQ ID NO:1. This synthetic polypeptide, as a chemically defined substance, replaces the complex and difficult-to-preserve natural proteins used in traditional double-antibody sandwich methods, thereby eliminating the inherent batch-to-batch variability and quality fluctuations of biological standards at the source. Based on this core reagent, the competitive ELISA detection system constructed in this embodiment achieves high specificity, high accuracy, and good reproducibility. Furthermore, because the synthetic polypeptide can be chemically synthesized at low cost and high throughput, the development cycle of the entire detection scheme is significantly shortened, and its economic efficiency is significantly improved.

[0042] The second aspect of this embodiment provides a kit for the quantitative detection of mouse statin, the kit comprising:

[0043] Includes a solid-phase support for the polypeptide of claim 1;

[0044] A series of concentration standards formulated from the polypeptide of claim 1;

[0045] An antibody capable of specifically binding to the polypeptide of claim 1.

[0046] This embodiment provides a detection kit comprising a solid-phase carrier coated with a specific synthetic polypeptide, a series of concentration standards formulated with the polypeptide, and an antibody. The kit achieves the technical effects of stable raw material sources, controllable quality, and simple operation, which not only significantly reduces the development threshold and production cost, but also ensures high accuracy and excellent repeatability of the detection results.

[0047] In some embodiments, the kit also contains enzyme-labeled streptavidin.

[0048] In some embodiments, the enzyme is horseradish peroxidase.

[0049] In some embodiments, the kit may also include a colorimetric solution and a stop solution.

[0050] The third aspect of this embodiment provides a method for quantitatively detecting inhibin in mouse blood samples, using a competitive ELISA method, the method comprising the following steps:

[0051] a. Providing a solid-phase support for coating the polypeptide of claim 1;

[0052] b. Add the series of concentration standard solutions and the pretreated mouse blood samples to be tested into different wells of the solid support, respectively;

[0053] c. Add an antibody against the polypeptide of claim 1, incubate to allow the inhibin in the sample to competitively bind to the antibody against the solid-coated polypeptide; discard the liquid, spin dry, add biotin-labeled antibody, and incubate.

[0054] d. After washing, add enzyme-labeled streptavidin and incubate;

[0055] e. After washing, add the color developing solution to carry out the color development reaction, and then add the stop solution to terminate the reaction;

[0056] f. Detect the absorbance value, and calculate the concentration of inhibin in the sample to be tested based on the standard curve plotted using the series of concentration standard solutions.

[0057] This embodiment provides a competitive ELISA detection method based on the specific synthetic peptide. This method achieves rapid and accurate quantification of mouse blood samples within one hour by using the core step of competitive binding of peptide-specific antibodies between the inhibin in the sample and the solid-phase coated peptide, combined with the subsequent signal amplification and color development system. It achieves the technical effects of simple operation, fast detection speed, high specificity and good repeatability.

[0058] In some embodiments, the colorimetric solution is a TMB colorimetric solution.

[0059] In some embodiments, the enzyme-labeled streptavidin is horseradish peroxidase-labeled streptavidin.

[0060] In some embodiments, the standard curve is plotted using a double logarithmic four-parameter logical curve fitting method.

[0061] In some embodiments, the calculated inhibin concentration is expressed in molar concentration units.

[0062] To better understand the technical solutions of the above embodiments, a more detailed embodiment 1 is provided for further explanation:

[0063] Example 1

[0064] 1. Reagent preparation

[0065] Microplates coated with α-subunit synthetic peptide of statin: 96-well microplates were coated with 5 μg / well.

[0066] Reference materials: Synthetic inhibin α subunit polypeptide, formulated into a series of reference materials with different μM concentrations.

[0067] Sample diluent: Used to dilute mouse blood samples.

[0068] Antibody: A specific antibody against the α subunit of inhibin.

[0069] Biotinylated anti-antibodies: Biotinylated antibodies against the above-mentioned antibodies from different species, such as biotinylated goat anti-rabbit IgG antibody.

[0070] Enzyme conjugates: Enzyme labels that are bound to biotin, such as horseradish peroxidase (HRP).

[0071] Colorimetric solution: TMB (3,3',5,5'-tetramethylbenzidine) colorimetric solution.

[0072] Termination solution: Usually a sulfuric acid solution, used to terminate the reaction.

[0073] Washing solution: A buffer solution used to wash the reaction plate.

[0074] 2. Sample preparation

[0075] Collect mouse blood samples, allow them to stand and coagulate, then centrifuge to separate serum, or treat with an anticoagulant to separate plasma.

[0076] Dilute the sample to the appropriate concentration according to the kit requirements. It is generally recommended to dilute it at least 10 times before adding it to the ELISA plate.

[0077] 3. Instrument Preparation

[0078] Microplate reader: Used to measure absorbance (OD value) at a wavelength of 450nm.

[0079] Incubator: Used for incubation at 37℃.

[0080] Pipettes and pipette tips: used for precise pipetting.

[0081] ELISA plate: used for the reaction.

[0082] Operating steps

[0083] Adding samples

[0084] Add 10 μL of sample to each well of the microplate, followed by 90 μL of sample diluent; add 100 μL of reference solution to each well, and add 100 μL of sample diluent to each blank well.

[0085] It is recommended to dilute the sample to be tested at least 10 times with sample diluent before adding it to the ELISA plate for testing, in order to reduce matrix effects.

[0086] 2. Warmth and Incubation

[0087] Place the ELISA plate in a 37°C incubator for 30 minutes.

[0088] 3. Discard the liquid

[0089] After incubation, discard the liquid, spin dry, and do not wash.

[0090] 4. Add biotin-labeled antibody

[0091] Add 100 μL of biotin-labeled antibody working solution to each well, mix well, and incubate at 37°C for 30 minutes.

[0092] 5. Washing the board

[0093] Wash the microplate thoroughly 4–6 times with 1x washing buffer, adding 350 μL of washing buffer to each well each time. Shake / soak for 1–2 minutes and then pat dry on filter paper.

[0094] 6. Add enzyme conjugates

[0095] Add 100 μL of enzyme conjugate working solution to each well, mix well, and incubate at 37°C for 15 minutes.

[0096] 7. Washing the board

[0097] Same as step 5, wash the ELISA plate thoroughly 4-6 times with 1x washing buffer.

[0098] 8.Add chromogenic solution

[0099] Add 100 μL of TMB colorimetric solution to each well, mix well, and then place in the dark at 37°C for 5–10 minutes. The specific colorimetric time depends on the colorimetric results.

[0100] 9. Add stop solution

[0101] Add 100 μL of stop solution to each well, mix well to stop the reaction, and immediately measure the optical density (OD value) of each well at a wavelength of 450 nm using an ELISA reader.

[0102] Result Calculation

[0103] 1. Plotting Standard Curves

[0104] Calculate the average OD value of the standard and sample replicates.

[0105] A standard curve is created by fitting a four-parameter logic curve on a logarithmic coordinate axis with the concentration of the standard sample as the x-axis and the OD value as the y-axis.

[0106] 2. Sample concentration calculation

[0107] Find the corresponding concentration on the standard curve based on the OD value of the sample.

[0108] If the OD value of the sample is higher than the upper limit of the standard curve, it should be diluted appropriately and retested. When calculating the sample concentration, the corresponding dilution factor should be multiplied to obtain the actual concentration of the sample.

[0109] Precautions

[0110] Incubation time and temperature: Strictly control incubation time and temperature to avoid insufficient or excessive incubation time and inaccurate temperature.

[0111] Pipette accuracy: Ensure accurate pipette calibration and avoid air bubbles during pipetting.

[0112] Thorough washing: The washing process must be thorough to avoid residual liquid affecting the results.

[0113] Color development time control: The color development time should not be too long to avoid the background value being too high.

[0114] Reagent storage: The reagents in the kit must be stored in accordance with the instructions to avoid reagent deterioration.

[0115] By following the steps described above, the α-subunit competitive ELISA method can be used to detect the level of inhibin in mouse blood.

[0116] The synthetic peptides coated in this application have a well-defined amino acid composition (FRPSQHTRSRQVTSA), a conserved sequence, and a purity of up to 95% or higher, which, together with the corresponding antibody, ensures high specificity.

[0117] This application uses a clearly defined synthetic peptide coupled with a carrier protein to prepare antibodies, which not only ensures the specificity of the antibodies but also fully guarantees their sensitivity. Combined with the existing biotin-streptavidin and TMB systems, the sensitivity reaches the pg level.

[0118] The reference curve for quantitative detection in this application is calibrated using synthetic peptides. The amino acid composition of the synthetic peptides (FRPSQHTRSRQVTSA) is well-defined, ensuring reliable results.

[0119] Specifically: SEQ ID NO:1 (FRPSQHTRSRQVTSA) is derived from: GenBank: ANJ16165.1. The corresponding CDS is "ASCGDEPDAGEAEEGLFTYVFRPSQHTRSRQVTSAQLWFHTGLDRQETTA TN".

[0120] Obtain the full sequence of inhibin-alpha through the NCBI website: MWPQLLLLLLAPRSGHGCQGPELDRELVL AKVRALFLDALGPPAVTGEGGDPGVRRLPRRHAVGGFMRRGSEPEEEDVSQAILFPATGARCGDEPAAGELAREAEEGLFTYVFRPSQHTRSRQVTSAQLWFHTGLDRQGMAAANSSGPLLDLLALSSRGPVAVPMSLGQAPPRWAVLHLAASALPLLTHPVLVLLLR CPLCSCSARPEATPFLVAHTRARPPSGGERARRSTAPLPWPWSPAALRLLQRPPEEPAVHADCHRASLNISFQELGWDRWIVHPPSFIFHYCHGGCGLPTLPNLPLSVPGAPPTPVQPLLLVPGAQPCCAALPGTMRSLRVRTTSDGGYSFKYETVPNLLTQHCACI.

[0121] like Figure 1 As shown, this paper presents an antigenic linear epitope prediction analysis of the full sequence of mouse α-inhibin subunit using bioinformatics software.

[0122] Three sequences, including FRPSQHTRSRQVTSA, were obtained for the preparation of an antibody against inhibin-alpha.

[0123] When the three antibodies were used in a mouse superovulation experiment, the sequence was found to be significantly more effective than the others, as shown in the test results. Figure 2 and Figure 3 As shown.

[0124] The detailed process is as follows: 24 hours after fertilization, the two-cell embryo and unfertilized egg cell are separated, and the embryonic development morphology, cell number, and other indicators are observed and recorded. Figure 2 and Figure 3 As shown in the figure, statistical analysis revealed that 150 two-cell embryos were obtained in the experimental group, with 93.29% of the fertilized eggs developing into two-cell embryos. In contrast, 81 two-cell embryos were obtained in the control group, with 82.7% of the fertilized eggs developing into two-cell embryos. The difference was highly statistically significant (P<0.01).

[0125] To investigate the relationship between serum inhibin levels and superovulation, microplates were directly coated with the FRPSQHTRSRQVTSA sequence. Inhibin in serum samples competitively binds to a specific antibody against the FRPSQHTRSRQVTSA sequence, thereby detecting the inhibin content in the serum sample.

[0126] Specifically: Calibration of the quantitative reference standard: The sequence "FRPSQHTRSRQVTSA" (15 residues) was calculated using the commonly used average amino acid residue mass (peptide residue mass), and the final peptide molecular weight was used as the average relative molecular mass of the whole peptide (g·mol⁻¹ / Da). The calculation steps and results are as follows:

[0127] Residue mass table used (commonly average residue mass, unit Da):

[0128] A(Ala)=71.08; R(Arg)=156.19; N(Asn)=114.10; D(Asp)=115.09; C(Cys)=103 .14; E(Glu)=129.12; Q(Gln)=128.13; G(Gly)=57.05; H(His)=137.14; I / L(Il e / Leu)=113.16; K(Lys)=128.17; M(Met)=131.19; F(Phe)=147.18; P(Pro)=97.12 ; S (Ser) = 87.08; T (Thr) = 101.11; W (Trp) = 186.21; Y (Tyr) = 163.18; V (Val) = 99.13.

[0129] List the mass of each residue in the sequence and add them together (in Da):

[0130] F=147.18; R=156.19; P=97.12; S=87.08; Q=128.13; H=137.14; T=101.11; R=1 56.19; S=87.08; R=156.19; Q=128.13; V=99.13; T=101.11; S=87.08; A=71.08.

[0131] The sum of residue masses = 1739.94 Da (derived by adding the above 15 items).

[0132] The molecular weight of a complete polypeptide (with N-terminal H and C-terminal OH) = the mass of the residues plus H2O.

[0133] =1739.94+18.01528=1757.9553Da.

[0134] Therefore, the average molecular weight (M) of the polypeptide is approximately 1757.96 g·mol⁻¹ (which can be written as 1757.96 Da to two decimal places).

[0135] The steps to convert 1 mg·mL⁻¹ to molar concentration are as follows:

[0136] 1 mg·mL⁻¹ = 1 g·L⁻¹.

[0137] Molar concentration (mol·L⁻¹) = Mass concentration (g·L⁻¹) ÷ Molecular weight (g·mol⁻¹) = 1.000 g·L⁻¹ ÷ 1757.9553 g·mol⁻¹ = 0.0005688427 mol·L⁻¹.

[0138] Replace with more commonly used units:

[0139] = 0.5688 mM (millomoles per liter);

[0140] = 568.84 μM (micromolar·L⁻¹).

[0141] The above sequences have clearly defined amino acid types and quantities, and the corresponding molecular weights can be accurately obtained. The synthesized high-purity (>90%) peptides can be used to prepare very accurate quantitative curves.

[0142] Calibration curve of the reference sample:

[0143] Table 1 Competitive ELISA Standard Curve Data

[0144]

[0145] Four-parameter Logistic curve fitting

[0146] Equation: y=(AD) / [1+(x / C)^B]+D.

[0147] A=3.47442; B=0.48951; C=75.76074; D=-0.08191; r^2=0.99780;

[0148] The number of data points is 8; the sum of squared residuals is 0.03144.

[0149] The data above show that the detection range of this method is from 0 (0.1 ng / ml) to 100 μg / ml (56.884 μM), spanning seven orders of magnitude. This is significantly superior to the detection range of existing double-antibody sandwich ELISA methods.

[0150] Specifically, the pretreated blood sample from the mouse to be tested is the mouse serum sample. 10 μL of serum is separated from the orbital blood collected from the mouse.

[0151] The biotin-labeled goat anti-rabbit antibody was purchased from Beyotime (product number: A0279-0.5ml).

[0152] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A polypeptide for detecting mouse inhibin, characterized by, The amino acid sequence of the polypeptide is shown as SEQ ID NO:

1.

2. A kit for quantitatively detecting mouse inhibin, characterized by comprising: The kit comprises: a solid phase carrier comprising the polypeptide of claim 1; a series of concentration standards prepared from the polypeptide of claim 1; an antibody capable of specifically binding to the polypeptide of claim 1.

3. The kit of claim 2, wherein The kit further comprises enzyme-labeled streptavidin.

4. The kit of claim 3, wherein The enzyme is horseradish peroxidase.

5. The kit of claim 2, wherein The kit further comprises color developing solution and termination solution.

6. A method for quantitatively detecting inhibin in mouse blood samples, characterized in that, The competitive ELISA method comprises the following steps: a. providing a solid phase carrier coated with the polypeptide of claim 1; b. adding a series of concentration standard solutions and pretreated blood samples of the mice to be tested into different wells of the solid phase carrier respectively; c. adding an antibody against the polypeptide of claim 1, incubating, allowing the inhibin in the sample to compete with the solid phase coated polypeptide for binding to the antibody; discarding the liquid, spinning dry, adding biotin-labeled antibody, incubating; d. after washing, adding enzyme-labeled streptavidin for incubation; e. after washing, adding color developing solution for color developing reaction, and then adding termination solution to terminate the reaction; f. detecting the absorbance value, and calculating the concentration of inhibin in the sample to be tested according to the standard curve drawn by the series of concentration standard solutions.

7. The method of claim 6, wherein, The color developing solution is TMB color developing solution.

8. The method of claim 6, wherein, The enzyme-labeled streptavidin is horseradish peroxidase-labeled streptavidin.

9. The method of claim 6, wherein, The standard curve is drawn by fitting a double logarithmic four-parameter logistic curve.

10. The method of claim 6, wherein, The calculated concentration of inhibin is expressed in units of substance concentration.

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