Kit for jointly detecting human serum Kisspeptin 1 based on ligand and antibody as well as preparation method and application of kit

Abstract

The invention relates to the technical field of immunodetection, in particular to a kit for jointly detecting human serum Kisspeptin 1 based on ligands and antibodies and a preparation method and application of the kit. The kit comprises a reagent R1, a reagent R2 and standard substance liquid; the method comprises the following steps: analyzing and screening a specific binding ligand of human Kisspeptin 1 to obtain a receptor protein Kiss-1receptor (Kisspeptin for short), coating magnetic beads by using the Kisspeptin 1R protein to obtain a reagent R1, and carrying out monoclonal labeling on acridinium ester on mouse anti-human Kisspeptin 1 to obtain a reagent R2. The kit prepared by the invention is combined with a chemiluminescence immunoassay instrument, the detection sensitivity is high, the specificity is good, the linear range is wide, the time is short, the operation is simple and convenient, the accurate detection of the Kisspeptin 1 in the human serum can be realized, and a reference is also provided for the clinical detection of the Kisspeptin 1 in the human serum.

Description

Technical Field

[0001] The present invention relates to the technical field of chemiluminescent immunoassay, and in particular to a kit for detecting human serum kisspeptin 1 based on a ligand and an antibody, a preparation method and an application thereof. Background Art

[0002] Human kisspeptin 1 is a peptide hormone in the human body, derived from the precursor protein kisspeptin encoded by the KISS1 gene. It is expressed in the hypothalamus, pancreas, placenta and ovary, and exerts biological effects by specifically binding to its receptor Kiss1R. During normal female pregnancy, the level of human kisspeptin 1 increases with the increase of pregnancy period. The expression level of human kisspeptin 1 is low in malignant gastrointestinal diseases, early spontaneous abortion, preeclampsia, polycystic ovary syndrome and other diseases, and high in gestational diabetes. Studies have shown that the level of human kisspeptin 1 is lower than normal in early spontaneous abortion diseases, and it continues to decrease with the increase of pregnancy period.

[0003] The kits currently on the market are all enzyme-linked immunosorbent assay kits for scientific research. They are based on the principle of antigen-antibody reaction and detect human kissin 1 through paired antibodies. It is necessary to prepare paired antibodies with different epitopes. The content of human kissin 1 in human serum is in the range of 1 to 50 ng / ml, which is relatively low. The molecular weight is about 6KD, which is relatively small compared to other proteins, and there are few antigenic epitopes. Therefore, it takes a lot of time to prepare and screen paired antibodies. This type of kit has the problems of low detection sensitivity, poor specificity detection, low detection accuracy, long detection time, and cumbersome operation. At present, there is no kit that can be used as a reference for clinical diagnosis. Therefore, it is urgent to prepare a highly sensitive and specific kit that can be used for the detection of human kissin 1 in human serum without cumbersome antibody pairing screening. Summary of the invention

[0004] To solve the above problems, the purpose of the present invention is to prepare a kit for detecting kisspeptin 1 in human serum based on a combination of a ligand and an antibody. The kit of the present invention has high sensitivity, strong specificity, short sample detection time, accurate results, avoids the cumbersome antibody pairing process, saves time and cost. The present invention can be applied to the detection of kisspeptin 1 in human serum.

[0005] To achieve the above object, the present invention adopts a kit for detecting human serum kisspeptin1 based on the combined use of ligand and antibody, comprising R1 reagent, R2 reagent and standard solution;

[0006] The R1 reagent is a magnetic bead solution coated with receptor protein;

[0007] The R2 reagent is an antibody labeling solution containing an acridinium ester label;

[0008] The standard solution is a synthetic human kisspeptin 1 antigen of different concentrations diluted with a PBS buffer solution containing 0.1% Proclin 300, 1% BSA, and 5% PEG 2000.

[0009] The present invention also provides a method for preparing a kit for detecting human serum kisspeptin 1 based on a combination of a ligand and an antibody, comprising the following steps:

[0010] Take the carboxyl magnetic bead solution, add carbodiimide and N-hydroxysuccinimide at the same time and mix for 1 minute to start activation;

[0011] Add human kisspeptin 1 receptor protein to the activated magnetic bead solution for overnight coating and coupling;

[0012] The coated magnetic beads were blocked with blocking solution at room temperature for 1 hour, washed twice, and then the preservation solution was added to obtain the magnetic bead coating solution, which is the R1 reagent;

[0013] Using mouse monoclonal antibody and acridinium ester for labeling and dialyzing to obtain an antibody labeling solution, the antibody labeling solution is the R2 reagent;

[0014] The standard solution was prepared by diluting different concentrations of human kisspeptin 1 synthetic antigen with PBS buffer containing 0.1% Proclin 300, 1% BSA, and 5% PEG 2000.

[0015] Among them, in the step of taking the carboxyl magnetic bead solution, adding carbodiimide and N-hydroxysuccinimide at the same time and mixing for 1 minute to start the activation:

[0016] The mass ratio of carboxyl magnetic beads, carbodiimide and N-hydroxysuccinimide is 1-10:1:1; the activation temperature is 2-37°C, and the activation time is 0.5-2 hours.

[0017] Wherein, in the step of adding human kisspeptin 1 receptor protein to the activated magnetic bead solution and performing overnight coating coupling:

[0018] The human kisspeptin 1 receptor protein was obtained by predicting and screening potential ligands of human kisspeptin 1; the mass ratio of human kisspeptin 1 receptor protein to magnetic beads was 0.1-1:1, and the coupling temperature was 2-37°C.

[0019] Among them, in the step of obtaining an antibody labeled solution after labeling with mouse monoclonal antibody and acridinium ester and dialyzing:

[0020] The labeling ratio of mouse monoclonal antibody and acridinium ester was 10-40:1, and the reaction was carried out at room temperature for 10 minutes before quenching and dialysis.

[0021] Wherein, in the step of preparing the standard solution by diluting different concentrations of human kisspeptin 1 synthetic antigen with PBS buffer containing 0.1% Proclin300, 1% BSA, and 5% PEG2000:

[0022] The dilution concentrations of the standard are: the standard is human kisspeptin 1 synthetic antigen, and the dilution concentrations of the standard are 0.1ng / ml, 1ng / ml, 10ng / ml, 20ng / ml, 30ng / ml, 40ng / ml, and 50ng / ml respectively.

[0023] The present invention also provides an application of a kit for detecting human serum kisspeptin 1 based on a combination of a ligand and an antibody, including an application in the field of detecting human serum kisspeptin 1.

[0024] The present invention can achieve the following beneficial effects:

[0025] The ligand used in the ligand and antibody pairing used in the kit for detecting human serum kisspeptin 1 based on the combined use of ligand and antibody provided by the present invention is human kisspeptin 1 receptor protein Kiss 1R, and the protein is obtained by a predictive screening method, thereby avoiding the screening process of paired antibodies in traditional reagents and shortening the raw material preparation time.

[0026] The kit for detecting human serum kisspeptin 1 based on the combined use of ligand and antibody provided by the present invention is used to detect the level of kisspeptin 1 in human serum, has high detection sensitivity, wide linear range, strong specificity, and short detection time, and can provide a reference for clinical detection of kisspeptin 1.

[0027] The kit for detecting human serum kisspeptin 1 based on the combined use of ligand and antibody provided by the present invention is used in conjunction with a fully automatic chemiluminescence instrument, thereby shortening the detection time, making the detection operation simpler, and achieving more accurate detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1The present invention is a flow chart of the preparation steps of the human kissin 1 detection kit.

[0030] Figure 2 The present invention is a schematic diagram of the amino acid sequence of the human kissin 1 receptor protein.

[0031] Figure 3 It is a schematic diagram of the concentration and luminescence value curve of the human kissin 1 synthetic antigen of the present invention.

[0032] Figure 4 It is a schematic diagram of the human kissin 1 level in spontaneous abortion samples at different gestational cycles of the present invention. DETAILED DESCRIPTION

[0033] The present invention provides a kit for detecting kisspeptin 1 in human serum based on a combination of a ligand and an antibody, comprising an R1 reagent, an R2 reagent and a standard solution;

[0034] The R1 reagent is a magnetic bead solution coated with receptor protein;

[0035] The R2 reagent is an antibody labeling solution containing an acridinium ester label;

[0036] The standard solution is a synthetic human kisspeptin 1 antigen of different concentrations diluted with a PBS buffer solution containing 0.1% Proclin 300, 1% BSA, and 5% PEG 2000.

[0037] See also Figure 1 The present invention provides a method for preparing a kit for detecting human serum kisspeptin 1 based on a ligand and an antibody, comprising the following steps:

[0038] S100: Take the carboxyl magnetic bead solution, add carbodiimide and N-hydroxysuccinimide at the same time and mix for 1 minute to start activation;

[0039] S200: Add human kisspeptin 1 receptor protein to the activated magnetic beads for overnight coating and coupling;

[0040] S300: taking a blocking solution to block the coated magnetic beads at room temperature for 1 hour, washing twice and then adding a preservation solution, the magnetic bead coating solution is the R1 reagent;

[0041] S400: labeling with mouse monoclonal antibody and acridinium ester and dialyzing to obtain an antibody labeling solution, which is the R2 reagent;

[0042] S500: diluting different concentrations of human kisspeptin 1 synthetic antigen in PBS buffer containing 0.1% Proclin 300, 1% BSA, and 5% PEG 2000 to prepare the standard solution;

[0043] S600: Combine the kit with a chemiluminescent immunoassay analyzer to detect kisspeptin 1 concentration in human serum.

[0044] The present invention also provides a method for preparing a kit for detecting human serum kisspeptin 1 based on a ligand and an antibody, including application in the field of detecting human kisspeptin 1.

[0045] In this embodiment, the chemical formula of carbodiimide is EDC, and the chemical formula of N-hydroxysuccinimide is NHS; first, take carboxyl magnetic bead solution, add carbodiimide (EDC) and N-hydroxysuccinimide (NHS) at the same time and mix for 1 minute to start activation; wherein, the mass ratio of carboxyl magnetic beads, EDC, and NHS is 1 to 10:1:1, the activation temperature is 2 to 37°C, and the activation time is 0.5 to 2 hours. Then add human kisspeptin 1 receptor protein to the activated magnetic bead solution for overnight coating and coupling; wherein, human kisspeptin 1 receptor protein is obtained by predicting and screening potential ligands of human kisspeptin 1, the mass ratio of human kisspeptin 1 receptor protein to magnetic beads is 0.1 to 1:1, and the coupling temperature is 2 to 37°C. Then take the blocking solution to block the coated magnetic beads at room temperature for 1 hour, then wash twice, and add the preservation solution. Then, mouse monoclonal antibody and acridinium ester were used for labeling and dialyzing; wherein, the labeling ratio of mouse monoclonal antibody and acridinium ester was 10-40:1, reacted at room temperature for 10 minutes, and then quenched and dialyzed. Finally, human kisspeptin 1 synthetic antigen was diluted with PBS buffer containing 0.1% Proclin300, 1% BSA, and 5% PEG2000; wherein, the standard product was human kisspeptin 1 synthetic antigen, and the dilution concentrations of the standard product were 0.1ng / ml, 1ng / ml, 10ng / ml, 20ng / ml, 30ng / ml, 40ng / ml, and 50ng / ml, respectively.

[0046] The present invention provides a kit for detecting human serum kisspeptin 1 based on a combination of ligand and antibody, which is used to detect the level of human kisspeptin 1 in human serum. The kit has high detection sensitivity, a wide linear range, strong specificity, and a short detection time, greatly reduces the antibody screening and pairing time, and can provide a reference for clinical detection of human kisspeptin 1.

[0047] The present invention will be described below in conjunction with the accompanying drawings and embodiments.

[0048] Example 1

[0049] (1) Prediction and screening of the final ligand of human kisspeptin 1.

[0050] HitPredict tool was used to predict potential interacting proteins of human kisspeptin 1, and ZDOCK software was used to screen the potential interacting proteins as the final ligands. The final ligand of human kisspeptin 1 was obtained as human kisspeptin 1 receptor protein, such as Figure 2 Shown is the amino acid sequence of the receptor protein. The human kisspeptin 1 receptor protein was expressed by a foreign company.

[0051] (2) Coupling of carboxyl magnetic beads.

[0052] Take 0.1ml (100mg / ml) carboxyl magnetic beads in an EP tube, mix with washing solution for 1 minute, discard the washing solution on the magnetic stand, add 0.5ml MES solution, weigh 2mg EDC and 2mg NHS, add to the magnetic bead solution and mix at room temperature for 1 minute, mix and activate at room temperature in the dark for 1 hour. Discard the supernatant on the magnetic stand, add 5mg human kisspeptin 1 receptor protein dissolved in cross-linking buffer, mix and react overnight at room temperature, discard the supernatant the next day, block with blocking solution at room temperature for 1 hour, discard the supernatant, add preservation solution and store at 4℃. The washing solution is a solution with a pH of 7.4 containing 0.85% NaCl, 2.69% Na2HPO4, 0.39% NaH2PO4, and 0.05% Tween-20, and MES is a 50mM MES solution with a pH of 6.0. The crosslinking buffer is a solution containing 2.95% Na2HPO4, 0.023% KH2PO4, 0.9% NaCl pH8.0. The preservation solution is a buffer containing 0.731% glycine, 0.075% anhydrous calcium chloride, 0.9% sodium chloride, 2.5% nystatin, 0.2% gelatin pH8.0. The blocking solution is a solution containing 2% BSA, 0.255% (V / V) ethanolamine pH8.0. Except for the percentage numbers in the brackets, all other percentages represent the mass fraction of each component in the total liquid.

[0053] (3) Acridinium ester labeling

[0054] Take 0.5 mg of anti-human kissin 1 mouse monoclonal antibody and dissolve it in 3 ml of pH 8.0 labeling solution to make antibody solution, take 1 mg of acridinium ester and dissolve it in 0.367 ml of DMSO to make acridinium ester solution, take 9.175 μl of acridinium ester solution and add it to the antibody solution, mix it and react it at room temperature in the dark for 10 minutes, add 1 ml of quenching solution and stir it at room temperature for 30 minutes, put the reaction solution in dialysate and dialyze it in a refrigerator at 4°C in the dark, change the dialysate every 4 hours, dialyze for 48 hours, collect the dialysate and store it at 4°C for later use. The labeling solution is a solution containing 2.95% Na2HPO4, 0.023% KH2PO4, 0.9% NaCl pH 8.0, and the quenching solution is a solution containing 2.95% Na2HPO4, 0.023% KH2PO4, 0.9% NaCl, 10% glycine pH 8.0. The dialysate is a PBS buffer solution containing 0.85% sodium chloride, 0.269% disodium hydrogen phosphate, and 0.039% sodium dihydrogen phosphate at pH 7.4. Except for the percentage numbers in brackets, all other percentages represent the mass fraction of each component in the total liquid.

[0055] Example 2

[0056] (1) Detection method of a kit for detecting kisspeptin 1 in human serum based on a combination of ligand and antibody

[0057] The detection procedure was established using the Kelaisi chemiluminescence immunoassay analyzer. 50 μl of magnetic bead coating solution and 50 μl of standard solution of each dilution concentration were incubated at 37°C for 15 minutes, washed 4 times with washing solution, 50 μl of acridinium ester labeling solution was added and incubated at 37°C for 10 minutes, washed 4 times with washing solution, and then 100 μl of pre-excitation solution and 100 μl of excitation solution were added to detect the luminescence value. The reaction curve of antigen concentration and luminescence value was obtained by parameter fitting method as shown in the figure. Figure 3 As shown. The washing liquid is a solution of 0.85% NaCl, 2.69% Na2HPO4, 0.39% NaH2PO4, and 0.05% (V / V) Tween-20 at pH 7.4, the stimulating liquid is a solution of 2% hexadecyltrimethylammonium chloride and 4% sodium hydroxide, and the pre-stimulating liquid is a 0.4% hydrogen peroxide solution at pH 1.5. Except for the percentage numbers in the brackets, the other percentages represent the mass fraction of each component in the total liquid.

[0058] (2) Detection linear range.

[0059] By detecting the luminescence values ​​of the above-mentioned standard samples with different concentrations and the luminescence curves of the detection concentrations, it was obtained that the linear correlation coefficient r was 0.997 and the linear range was 0.1-40 ng / ml.

[0060] (3) Detection sensitivity

[0061] The 0 ng / ml standard solution was tested repeatedly 20 times on the chemiluminescence detection instrument to obtain the luminescence value and calculate its average and standard deviation. The average value plus 2 times the standard deviation was substituted into the above standard concentration and luminescence value curve formula to obtain a concentration value of 0.63 ng / ml, which is the detection sensitivity.

[0062] (4) Detection repeatability

[0063] The three standard solutions of 10 ng / ml, 20 ng / ml and 40 ng / ml were tested repeatedly 10 times on the chemiluminescence instrument, and the mean and standard deviation of the three standard solutions were calculated. Their linear differences CV were calculated by dividing the standard deviation by the mean, which were 6.2%, 5.7% and 4.5%, respectively, all less than 10%.

[0064] (5) Specificity test

[0065] A mixed serum was prepared with negative serum at a mass fraction of 15% for hemoglobin, bilirubin, triglyceride and vitamin C. The luminescence values ​​of the mixed serum and the corresponding negative serum of these substances were tested on a chemiluminometer, and their relative deviations were calculated. The results were all within 10%, which met the requirements.

[0066] Example 3

[0067] (1) Coupling of carboxyl magnetic beads.

[0068] Take 0.1ml (100mg / ml) carboxyl magnetic beads in an EP tube, mix with washing solution for 1 minute, discard the washing solution on the magnetic stand, add 0.5ml MES solution, weigh 1mg EDC and 1mg NHS, add to the magnetic bead solution and mix at room temperature for 1 minute, and activate at 37℃ in the dark for 0.5 hours. Discard the supernatant on the magnetic stand, add 1mg human kisspeptin 1 receptor protein that has been dialyzed and dissolved in cross-linking buffer, mix and react overnight at 37℃, discard the supernatant the next day, block with blocking solution at room temperature for 1 hour, discard the supernatant, add preservation solution and store at 4℃. The washing solution is a solution with a pH of 7.4 containing 0.85% NaCl, 2.69% Na2HPO4, 0.39% NaH2PO4, and 0.05% Tween-20, and MES is a 50mM MES solution with a pH of 6.0. The crosslinking buffer is a solution containing 2.95% Na2HPO4, 0.023% KH2PO4, 0.9% NaCl pH8.0. The preservation solution is a buffer containing 0.731% glycine, 0.075% anhydrous calcium chloride, 0.9% sodium chloride, 2.5% nystatin, 0.2% gelatin pH8.0. The blocking solution is a solution containing 2% BSA, 0.255% (V / V) ethanolamine pH8.0. Except for the percentage numbers in the brackets, all other percentages represent the mass fraction of each component in the total liquid.

[0069] (2) Acridinium ester labeling

[0070] Take 0.5 mg of anti-human kissin 1 mouse monoclonal antibody and dissolve it in 3 ml of pH 8.0 labeling solution to make antibody solution, take 1 mg of acridinium ester and dissolve it in 0.367 ml of DMSO to make acridinium ester solution, take 4.588 μl of acridinium ester solution and add it to the antibody solution, mix it and react it at room temperature in the dark for 10 minutes, add 1 ml of quenching solution and stir it at room temperature for 30 minutes, put the reaction solution in dialysate and dialyze it in a refrigerator at 4°C in the dark, change the dialysate every 4 hours, dialyze for 48 hours, collect the dialysate and store it at 4°C for later use. The labeling solution is a solution containing 2.95% Na2HPO4, 0.023% KH2PO4, 0.9% NaCl pH 8.0, and the quenching solution is a solution containing 2.95% Na2HPO4, 0.023% KH2PO4, 0.9% NaCl, 10% glycine pH 8.0. The dialysate is a PBS buffer solution containing 0.85% sodium chloride, 0.269% disodium hydrogen phosphate, and 0.039% sodium dihydrogen phosphate at pH 7.4. Except for the percentage numbers in brackets, all other percentages represent the mass fraction of each component in the total liquid.

[0071] Example 4

[0072] (1) Coupling of carboxyl magnetic beads.

[0073] Take 0.1ml (100mg / ml) carboxyl magnetic beads in an EP tube, mix with washing solution for 1 minute, discard the washing solution on the magnetic stand, add 0.5ml MES solution, weigh 10mg EDC and 10mg NHS, add to the magnetic bead solution and mix at room temperature for 1 minute, and activate at 2℃ in the dark for 2 hours. Discard the supernatant on the magnetic stand, add 10mg human kisspeptin 1 receptor protein that has been dialyzed and dissolved in cross-linking buffer, mix and react overnight at 2℃, discard the supernatant the next day, block with blocking solution at room temperature for 1 hour, discard the supernatant, add the preservation solution and store at 4℃. The washing solution is a solution with a pH of 7.4 containing 0.85% NaCl, 2.69% Na2HPO4, 0.39% NaH2PO4, and 0.05% Tween-20, and MES is a 50mM MES solution with a pH of 6.0. The crosslinking buffer is a solution containing 2.95% Na2HPO4, 0.023% KH2PO4, 0.9% NaCl pH8.0. The preservation solution is a buffer containing 0.731% glycine, 0.075% anhydrous calcium chloride, 0.9% sodium chloride, 2.5% nystatin, 0.2% gelatin pH8.0. The blocking solution is a solution containing 2% BSA, 0.255% (V / V) ethanolamine pH8.0. Except for the percentage numbers in the brackets, all other percentages represent the mass fraction of each component in the total liquid.

[0074] (2) Acridinium ester labeling

[0075] Take 0.5 mg of anti-human kissin 1 mouse monoclonal antibody and dissolve it in 3 ml of pH 8.0 labeling solution to make antibody solution, take 1 mg of acridinium ester and dissolve it in 0.367 ml of DMSO to make acridinium ester solution, take 18.35 μl of acridinium ester solution and add it to the antibody solution, mix it and react it at room temperature in the dark for 10 minutes, add 1 ml of quenching solution and stir it at room temperature for 30 minutes, put the reaction solution in dialysate and dialyze it in a refrigerator at 4°C in the dark, change the dialysate every 4 hours, dialyze for 48 hours, collect the dialysate and store it at 4°C for later use. The labeling solution is a solution containing 2.95% Na2HPO4, 0.023% KH2PO4, 0.9% NaCl pH 8.0, and the quenching solution is a solution containing 2.95% Na2HPO4, 0.023% KH2PO4, 0.9% NaCl, 10% glycine pH 8.0. The dialysate is a PBS buffer solution containing 0.85% sodium chloride, 0.269% disodium hydrogen phosphate, and 0.039% sodium dihydrogen phosphate at pH 7.4. Except for the percentage numbers in brackets, all other percentages represent the mass fraction of each component in the total liquid.

[0076] Example 5

[0077] The test kit was used to detect 10 serum samples of pregnant women with spontaneous abortion in the 6th, 9th and 12th weeks on a chemiluminescence instrument. The concentration of human serotonin 1 in the samples was obtained by detecting the concentration on the chemiluminescence platform according to the detection method of Example 2. The average value and standard deviation of different gestational cycles were calculated as follows: Figure 4 As shown, the results showed that the level of human kisspeptin 1 in the serum of spontaneous abortion patients decreased with the increase of gestational age, which is consistent with the results of the current study.

[0078] The present invention provides a kit for detecting human serum kisspeptin 1 based on the combined use of a ligand and an antibody. The kit has high detection sensitivity, strong specificity, and a wide linear range, avoids the complicated screening process of paired antibodies in traditional antigen-antibody detection, shortens the detection time, and provides accurate and reliable detection results, which can provide a reference for clinical diagnosis and detection of human kisspeptin 1.

[0079] What is disclosed above is only a preferred embodiment of the present invention, and it certainly cannot be used to limit the scope of rights of the present invention. Ordinary technicians in this field can understand that all or part of the processes of the above embodiment and equivalent changes made according to the claims of the present invention still fall within the scope of the invention.

Claims

1. A kit for detecting kisspeptin 1 in human serum based on a combination of ligand and antibody, characterized in that: Includes R1 reagent, R2 reagent and standard solution; The R1 reagent is a magnetic bead solution coated with receptor protein; The R2 reagent is an antibody labeling solution containing an acridinium ester label; The standard solution is a synthetic human kisspeptin 1 antigen of different concentrations diluted with a PBS buffer solution containing 0.1% Proclin 300, 1% BSA, and 5% PEG 2000.

2. A method for preparing a kit for detecting human serum kisspeptin 1 based on a combination of ligand and antibody, comprising preparing a kit for detecting human serum kisspeptin 1 based on a combination of ligand and antibody as claimed in claim 1, characterized in that: The steps include: Take the carboxyl magnetic bead solution, add carbodiimide and N-hydroxysuccinimide at the same time and mix for 1 minute to start activation; Add human kisspeptin 1 receptor protein to the activated magnetic beads for overnight coating and coupling; The coated magnetic beads were blocked with blocking solution at room temperature for 1 hour, washed twice, and then the preservation solution was added to obtain the magnetic bead coating solution, which is the R1 reagent; Using mouse monoclonal antibody and acridinium ester for labeling and dialyzing to obtain an antibody labeling solution, the antibody labeling solution is the R2 reagent; The standard solution was prepared by diluting different concentrations of human kisspeptin 1 synthetic antigen with PBS buffer containing 0.1% Proclin 300, 1% BSA, and 5% PEG 2000.

3. The method for preparing a kit for detecting human serum kisspeptin 1 based on a ligand and an antibody as claimed in claim 2, characterized in that: In the step of taking the carboxyl magnetic bead solution, add carbodiimide and N-hydroxysuccinimide at the same time and mix for 1 minute to start the activation step: The mass ratio of carboxyl magnetic beads, carbodiimide and N-hydroxysuccinimide is 1-10:1:1; the activation temperature is 2-37°C, and the activation time is 0.5-2 hours.

4. The method for preparing a kit for detecting human serum kisspeptin 1 based on a ligand and an antibody as claimed in claim 2, characterized in that: In the step of adding human kisspeptin 1 receptor protein to the activated magnetic beads for overnight coating and coupling: The human kisspeptin 1 receptor protein was obtained by predicting and screening potential ligands of human kisspeptin 1; the mass ratio of human kisspeptin 1 receptor protein to magnetic beads was 0.1-1:1, and the coupling temperature was 2-37°C.

5. The method for preparing a kit for detecting human serum kisspeptin 1 based on a ligand and an antibody as claimed in claim 2, characterized in that: In the step of obtaining an antibody labeled solution after labeling with a mouse monoclonal antibody and acridinium ester and dialysis: The labeling ratio of mouse monoclonal antibody and acridinium ester was 10-40:1, and the reaction was carried out at room temperature for 10 minutes before quenching and dialysis.

6. The method for preparing a kit for detecting human serum kisspeptin 1 based on a ligand and an antibody as claimed in claim 2, characterized in that: In the step of preparing the standard solution by diluting different concentrations of human kisspeptin 1 synthetic antigen in PBS buffer containing 0.1% Proclin 300, 1% BSA, and 5% PEG 2000: The dilution concentrations of the standard are: the standard is human kisspeptin 1 synthetic antigen, and the dilution concentrations of the standard are 0.1ng / ml, 1ng / ml, 10ng / ml, 20ng / ml, 30ng / ml, 40ng / ml, and 50ng / ml respectively.

7. A kit for detecting kisspeptin 1 in human serum based on a combination of ligand and antibody, characterized in that: Including applications in the field of detecting kisspeptin 1 in human serum.

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