WO2013071702A1

WO2013071702A1 - Preeclampsia detecting kit using adipsin as indicator and its preparing method

Info

Publication number: WO2013071702A1
Application number: PCT/CN2012/070010
Authority: WO
Inventors: 胡怀忠
Original assignee: 成都创宜生物科技有限公司
Priority date: 2011-11-17
Filing date: 2012-01-04
Publication date: 2013-05-23
Also published as: CN102426243B; CN102426243A

Abstract

A preeclampsia detecting kit using Adipsin as indicator, comprises porous plate coated with anti-human Adipsin monoclonal antibody, anti-human Adipsin monoclonal antibody detecting solution marked by biotin, avidin horseradish peroxidase combined with anti-human Adipsin monoclonal antibody marked by biotin, chromogenic substrate 3',3',5,5'-tetramethylbenzidine and Adipsin protein standard substance. The preparing method of the detecting kit comprises:（1）preparing the porous plate coated with anti-human Adipsin monoclonal antibody; (2) preparing the anti-human Adipsin monoclonal antibody detecting solution marked by biotin;(3) preparing the avidin horseradish peroxidase combined with anti-human Adipsin monoclonal antibody marked by biotin;(4) preparing the chromogenic substrate 3',3',5,5'-tetramethylbenzidine and Adipsin protein standard substance.

Description

Preeclampsia test kit using Adipsin as a detection index and preparation method The application request is submitted to the Chinese Patent Office on November 17, 2011, and the application number is

201110365299.7, the priority of the present invention is the priority of the Chinese Patent Application, the disclosure of which is incorporated herein by reference. Technical field

The invention belongs to the field of medical kits, and particularly relates to a kit for detecting preeclampsia (PE) of a pregnant woman and a preparation method thereof. Background technique

Preeclampsia (PE) is an obstetric complication that occurs after 20 weeks of gestation and is one of the leading causes of death in pregnant women and perinatal children. The prevalence of preeclampsia in pregnant women worldwide is 2-8%, and the incidence rate in pregnant women in China is 10%. Severe preeclampsia patients may have severe clinical manifestations such as hypertension, edema and severe proteinuria. They are also prone to complicated pulmonary edema, renal failure, HELLP syndrome, etc., long-term cardiovascular disease, kidney disease and metabolism. The risk of disease is also significantly increased. For the fetus, the smaller the gestational age, the lower the survival rate, the premature infants before 32 weeks of pregnancy, the mortality rate is as high as 50%, and the chance of serious complications such as asphyxia, intracranial hemorrhage and infection in neonates is 5-10%. The incidence of metabolic diseases and cardiovascular diseases in the long-term is also increasing, which seriously affects the health of the offspring, and brings a heavy psychological and health economic burden to the family and society.

The prior art clinical diagnosis of pre-eclampsia needs to meet the following three criteria: (1) maternal private complaints have clinical symptoms such as headache and dizziness; (2) The blood pressure of pregnant women meets the diagnostic criteria of hypertension, contraction by clinician Pressure/diastolic blood pressure ≥140/90 mmHg; (3) 24-hour urinary albumin quantitative test results were 24-hour proteinuria ≥0.3 g/day. The 24-hour urinary albumin quantitative test requires the collection of urine samples for 24 hours. This method of collecting samples is cumbersome and time consuming.

Adipsin (also known as factor D, or complement factor D, Chinese name "complement factor D") is a serine protease produced by the moon's fat cells, which is present in circulating blood and is closely related to the body's fat metabolism. Adipsin levels in circulating blood were stable in healthy pregnant women and pregnant women with preeclampsia, and there was no significant difference between the two groups. However, due to vascular endothelial damage in pregnant women with pre-eclampsia, renal vascular endothelial cell damage causes impaired renal permeability and recovery, resulting in High concentrations of Adipsin appear in urine samples from pregnant women with pre-eclampsia, so Adipsin can be used as a diagnostic and predictive indicator for pre-eclampsia based on this research basis. The Adipsin content in urine samples has not been reported as a test indicator for preeclampsia, and the pre-eclampsia test kit using the Adipsin content in urine samples as a detection index and its preparation method have not been reported. Summary of the invention

The object of the present invention is to provide a novel pre-eclampsia detection kit using the Adipsin contained in a urine sample as a detection index and a preparation method thereof, and to reduce the detection cost and reduce the pain of the patient by using the pre-eclampsia detection procedure. .

The inventors of the present patent application found through experiments that: Adipsin is stable in circulating blood of healthy pregnant women and pregnant women with preeclampsia, and the content of Adipsin in the blood of preeclampsia group is 2929.37±814.08 ng/mL, healthy pregnancy group The content of Adipsin in the blood was 2359.76±667.63 ng/mL, and there was no significant difference between the two groups. However, due to vascular endothelial injury in pregnant women with preeclampsia, renal vascular endothelial cell damage causes impaired renal permeability and recovery, resulting in high concentrations of Adipsin in urine samples from pregnant women with preeclampsia. , protein chip screening experiments found: the pre-eclampsia women diagnosed compared with the pre-eclampsia women diagnosed (the age of the two groups maternal age with the gestational age, no underlying disease, no other pregnancy complications), in the urine sample The concentration of Adipsin was significantly different. Quantitative study found that the content of Adipsin in the urine sample of pre-eclampsia group was 349.44±557.10 ng/mL, and the content of Adipsin in the urine sample of healthy pregnancy group was 8.69±6.34 ng/mL, due to the preeclampsia group. There is a significant difference in the content of Adipsin in the urine sample of the healthy pregnancy group. Therefore, the content of Adipsin in the urine sample can be used as a test index for judging whether a pregnant woman has pre-eclampsia.

The pre-eclampsia detection kit using Adipsin as a detection index according to the present invention comprises a multi-well plate coated with an anti-human Adipsin monoclonal antibody, a biotin-labeled anti-human Adipsin monoclonal antibody (Biotin-AdipsinAb) detection solution, and Biotinylated anti-human Adipsin monoclonal antibody

(Biotin-AdipsinAb) Binding of avidin - horseradish peroxidase, chromogenic substrate 3', 3', 5, 5'-tetradecyl benzidine and Adipsin protein standards.

Experiments have shown that the above kit contains a multi-well plate coated with an anti-human Adipsin monoclonal antibody, preferably 0.9 to 1 g per anti-human Adipsin monoclonal antibody. In the above kit, the biotin-labeled anti-human Adipsin monoclonal antibody

(Biotin-AdipsinAb) assay consists mainly of biotinylated anti-human Adipsin monoclonal antibody (Biotin-AdipsinAb), bovine serum albumin, glycerol and phosphate buffer, biotinylated anti-human Adipsin monoclonal antibody (Biotin The concentration of -AdipsinAb is 24 mg/mL, the concentration of bovine serum albumin is 1.5 g ~ 3 g/100 mL, and the concentration of glycerol is 50 mL/100 mL.

The biotinylated anti-human Adipsin monoclonal antibody (Biotin-AdipsinAb) is composed of biotinyl-N-hydroxysuccinimide ester and anti-human Adipsin monoclonal antibody, acyl-N-hydroxysuccinimide The mass ratio of the amine ester to the anti-human Adipsin monoclonal antibody is 0.5 to 1:7.

For the convenience of the user, the kit of the present invention may also be provided with a sample diluent which is in a phosphate buffer solution (PBS) or a concentration of 0.01 mol/L at a concentration of 0.01 mol/L to 0.02 mol/L. The concentration of bovine serum albumin was lg ~ 2 g / 100 mL in 0.02 mol / L trishydroxyl amino oxane buffer (Tris buffer, TBS) with bovine serum albumin.

The preparation method of the pre-eclampsia detection kit using Adipsin as the detection index of the present invention has the following steps:

(1) Preparation of a multi-well plate coated with an anti-human Adipsin monoclonal antibody

The anti-human Adipsin monoclonal antibody was diluted to a concentration of 10 / mL with a carbonate buffer of pH = 8 ~ 10, and anhydrous sterol, anhydrous sterol was added to the anti-human Adipsin monoclonal antibody dilution. The dosage is limited to a concentration of 3 mL/100 mL, and then the sterol-containing anti-human Adipsin monoclonal antibody dilution is added to each well of the multiwell plate and coated at 4 ° C for at least 12 hours; After the expiration of the time, the bovine serum albumin solution at a concentration of 1 g/100 mL was added to each well of the multiwell plate and blocked at 37 ° C for a reaction time of at least 1 hour; after the blocking reaction was completed, pH = The porous plate was washed with phosphate buffer of 7.2. When the unreacted material on the porous plate was removed, a multi-well plate coated with anti-human Adipsin monoclonal antibody was obtained, and the storage temperature was 4 ° C;

(2) Preparation of biotin-labeled anti-human Adipsin monoclonal antibody (Biotin-AdipsinAb) test solution

1 using hydrazine, hydrazine-dihydrazinyl amide as solvent, biotinyl-hydrazine hydroxysuccinimide ester as solute to prepare a concentration of 50 g / L of acyl-N hydroxysuccinimide ester solution, to pH = 9.6 carbonate buffer is the solvent, anti-human Adipsin monoclonal antibody is the solute and the concentration is 24 mg/mL. a mixture of the acyl-N-hydroxysuccinimide ester solution and the anti-human Adipsin monoclonal antibody solution is reacted at room temperature for at least 4 hours under stirring to obtain a biotin-labeled antibody. Human Adipsin monoclonal antibody (Biotin-AdipsinAb); After the reaction, the reaction solution containing biotin-labeled anti-human Adipsin monoclonal antibody (Biotin-AdipsinAb) was loaded into a dialysis bag with phosphate buffer pH 9.2. Dialysis is carried out at 4 ° C for at least 12 hours, during which the dialysate is exchanged at least 3 times; followed by addition of bovine serum albumin to the reaction solution after dialysis, the amount of bovine serum albumin added thereto The concentration is limited to 3 g ~ 6 g / 100 mL;

2 Biotin-labeled anti-human Adipsin monoclonal antibody prepared in step 1

(Biotin-AdipsinAb) and bovine serum albumin dialysis reaction solution and glycerol in a volume ratio of 1: 1 , mixed at room temperature to form biotin-labeled anti-human Adipsin monoclonal antibody

(Biotin-AdipsinAb) test solution, the storage temperature is - 20 ° C;

(3) Equipped with avidin-horseradish peroxidase, chromogenic substrate 3', 3', 5,5'-tetradecylbenzidine and Adipsin protein standards. The avidin-horseradish peroxidase, 3',3',5,5'-tetradecylbenzidine and Adip sin protein standards are commercially available and can be purchased directly from the market.

In the method of the present invention, when preparing a multi-well plate coated with an anti-human Adipsin monoclonal antibody, the anti-human Adipsin monoclonal antibody dilution is preferably added in an amount of 100 μΙ7 well, and the concentration is 1 g/100 mL. The bovine serum albumin solution was added in an amount of 120 μΙ 7 wells.

In the method of the present invention, a biotin-labeled anti-human Adipsin monoclonal antibody is prepared.

(Biotin-AdipsinAb), preferably, the acyl-N hydroxysuccinimide ester solution is obtained by mass ratio of acyl-N hydroxysuccinimide ester to anti-human Adipsin monoclonal antibody = 0.5-1:7 A mixture is formed with the anti-human Adipsin monoclonal antibody solution.

For convenience of the user, the method of the present invention is further provided with a sample diluent which is in a phosphate buffer solution (PBS) at a concentration of 0.01 mol/L to 0.02 mol/L or a concentration of 0.01 mol/L to 0.02 mol. /L trihydroxydecylaminodecane buffer (Tris buffer, TBS) is prepared by adding bovine serum albumin, and the bovine serum albumin is added in an amount of lg ~ 2 g / 100 mL. limit.

The invention has the following beneficial effects:

(1) The present invention provides a kit for detecting a pre-eclampsia with a new detection index and a preparation method thereof.

(2) Quantitative detection of Adipsin in urine samples of pregnant women using the kit of the present invention The exact content.

(3) Using the kit of the present invention to detect and diagnose pre-eclampsia women and healthy pregnant women (see Example 2), the test results show that: the sensitivity of the kit is 90%, and the specificity is about 80%. Therefore, the accuracy of pre-eclampsia detection can be improved.

(4) Since the test sample is a urine sample of a pregnant woman, it is a non-invasive test.

The kit of the invention is suitable for all pregnant women. DRAWINGS

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic view showing the structure of a porous plate in a pre-eclampsia detection kit using Adipsin as a detection index of the present invention.

Figure 2 shows the A450 value - Adipsin concentration standard curve. detailed description

In order to further understand the invention, the preferred embodiments of the present invention are described in the accompanying drawings.

The effects of the present invention are described below by way of specific examples, but the scope of the present invention is not limited by the following examples. Hereinafter, the pre-eclampsia detection kit using Adipsin as an indicator of detection according to the present invention, a preparation method thereof and a use method thereof will be further described by way of examples.

The sources of materials referred to in the following examples are as follows:

Mouse anti-human Adipsin monoclonal antibody, purchased from American R&D Company or RayBiotec Company; Adipsin protein standard product, purchased from American R&D Company or RayBiotec Company;

Carbonate buffer (CB), purchased from the US sigma company;

Phosphate buffer (PBS), purchased from the US sigma company;

Trihydroxydecylaminodecane buffer (Tris buffer, TBS), purchased from the US sigma company; bovine serum albumin, purchased from the US sigma company;

Ν,Ν-dimercaptoamide, purchased from American sigma company; Acyl-N-hydroxysuccinimide ester, purchased from sigma, USA;

The specification for glycerol is analytical alcohol, purchased from the US sigma company;

Horseradish peroxidase, purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.;

3',3',5,5'-tetradecylbenzidine, purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.;

The perforated plate was a 96-well multi-well plate available from Nunc, Denmark, and its shape and construction are shown in Fig. 1. Example 1

In this embodiment, the following process steps are used to prepare a pre-eclampsia test kit using Adipsin as an indicator:

(1) Preparation of a multi-well plate coated with a mouse anti-human Adipsin monoclonal antibody

The mouse anti-human Adipsin monoclonal antibody was diluted to a concentration of 10 /mL with a pH = 9 carbonate buffer, and anhydrous sterol was added to the antibody dilution. The amount of anhydrous sterol was reached at the concentration. Limiting to 3 mL/100 mL, the sterol-containing mouse anti-human Adipsin monoclonal antibody dilution was added to each well of a 96-well plate in an amount of 100 μΙ 7 well, and coated at 4 ° C for 12 hours; After the expiration of the time, the bovine serum albumin solution at a concentration of 1 g/100 mL was added to each well of the multiwell plate in an amount of 120 μΙ 7 wells and blocked at 37 ° C for 1 hour; after the blocking reaction was completed The porous plate was washed with a phosphate buffer solution having a pH of 7.2 and a concentration of 0.05 mol/L. When the unreacted material on the porous plate was removed, a porous plate coated with a mouse anti-human Adipsin monoclonal antibody was obtained, and then It is stored in a refrigerator at 4 °C for later use;

(2) Preparation of biotinylated mouse anti-human Adipsin monoclonal antibody (Biotin-AdipsinAb) assay solution

1 using hydrazine, hydrazine-dihydrazinamide as solvent, biotinyl-hydrazino hydroxysuccinimide as solute to prepare a concentration of 50 g / L of acyl-N hydroxysuccinimide ester solution, to pH = 9.6, a 1 mol/L carbonate buffer as a solvent, a mouse anti-human Adipsin monoclonal antibody as a solute, a concentration of 24 mg/mL of a mouse anti-human Adipsin monoclonal antibody solution, according to acyl-N-hydroxybutyrate a mass ratio of the diimide ester to the mouse anti-human Adipsin monoclonal antibody = 1:7 to form a mixture of the acyl-N-hydroxysuccinimide ester solution and the mouse anti-human Adipsin monoclonal antibody solution a mixture of the acyl-N-hydroxysuccinimide ester solution and the mouse anti-human Adipsin monoclonal antibody solution was reacted at room temperature for 4 hours under stirring (magnetic stirrer, stirring speed 200 rpm). Biotinylated mouse anti-human Adipsin Monoclonal antibody (Biotin-AdipsinAb); after the reaction, the reaction solution containing biotinylated mouse anti-human Adipsin monoclonal antibody (Biotin-AdipsinAb) was loaded into a dialysis bag with pH = 9.2 and a concentration of 0.05 mol/ The phosphate buffer of L was dialyzed at 4 ° C for 12 hours, during which the dialysate was changed 3 times; then the bovine serum albumin was added to the reaction solution after dialysis, the bovine serum albumin The amount added is limited to a concentration of 4 g/100 mL;

2 Biotin-labeled mouse anti-human Adipsin monoclonal antibody prepared in step 1

(Biotin-AdipsinAb) and bovine serum albumin dialysis reaction solution and glycerol in a volume ratio of 1: 1 , mixed at room temperature to form biotinylated mouse anti-human Adipsin monoclonal antibody

(Biotin-AdipsinAb) test solution, the storage temperature is - 20 ° C;

(3) Each kit is equipped with avidin-horseradish peroxidase 12 mL, chromogenic substrate 3',3',5,5'-tetradecylbenzidine 12 mL, Adipsin protein standard (Adipsin) The concentration is 10 ng/mL) 1 mL and the sample diluent is 30 mL. The sample dilution is added to the bovine serum white in a concentration of 0.02 mol/L Tris buffer (Tris buffer, TBS). The protein is prepared by adding the bovine serum albumin to a concentration of 2 g/100 mL; the avidin-horseradish peroxidase and the biotin-labeled mouse anti-human Adipsin monoclonal antibody The volume ratio of the test solution was 1:1. Example 2

In this example, the sample to be tested is tested by the pre-eclampsia test kit prepared in Example 1.

1. Sample to be tested

(1) Standard preparation

The Adipsin protein standard equipped in the kit described in Example 1 was set to the standard containing the highest concentration of Adipsin, and was designated as Standard 1, and the concentration of Adipsin was 6 ng/mL, and then the kit described in Example 1 was used. Diluted the Adipsin protein standard into a sample dilution

Adipsin concentrations of 2000 pg / mL, 666.7 pg / mL, 222.2 pg / mL, 74.07 pg / mL, 24.69 pg / mL, 8.23 pg / mL standards, named as standard 2, standard 3, standard 4 , Standard 5, Standard 6 and Standard 7 have a total of 7 standards.

(2) sample

Taking the pregnant women and hospitalized pregnant women of West China Second Hospital of Sichuan University as the experimental subjects, the experimental subjects were divided into the eclampsia group and the healthy control group according to the test results of the existing detection methods, and the urine samples of the two groups of pregnant women were collected. For the sample, a total of 40 samples, of which samples 1-20 were taken from the eclampsia group, and samples 21-40 were taken from the healthy control group.

(3) Blank control

The sample dilution provided in the kit of Example 1 was a blank control.

2, sample testing

(1) All reagents and samples to be tested are slowly balanced to room temperature (18 ° C ~ 25 ° C) before testing;

(2) Add the standard, blank control and sample prepared in step 1 to the corresponding wells on a 96-well plate coated with mouse anti-human Adipsin monoclonal antibody, each standard, blank control, sample Multiple wells were set up; the loading amount in each well was 100 L. The 96-well plate was covered with a film, and the 96-well plate was placed on a shaker and incubated at room temperature for 2.5 hours under shaking at a shaking speed of 100 rpm.

(3) After the incubation time expires, the liquid in each sample well is drained, and a cleaning procedure is set on the automatic washer, with a 400 μΙ 7-well wash (concentration of 0.02 mol/L phosphate buffer or a concentration of 0.02 mol/ L Tris buffer) Wash each well twice. For each wash, wash the solution for 10 seconds to 15 seconds. After cleaning, turn the 96-well plate to make each well. The mouth is down, and tapping on the absorbent filter paper completely removes the residual washing liquid in each sample well.

(4) 100 μΐ of biotin-labeled mouse anti-human Adipsin monoclonal antibody (Biotin-AdipsinAb) detection solution was added to each well of a 96-well plate, and then the 96-well plate was covered with a film, and The 96-well plate was placed on a shaker and incubated at room temperature for 1.5 hours at a shaking state, and the shaking speed was 100 rpm. After the incubation time expired, the liquid in each of the sample wells was exhausted, and step (3) was followed. The method cleans the 96-well plate.

(5) adding 100 μL of avidin-horseradish peroxidase to each of the wells of the 96-well plate, and then covering the 96-well plate with a film, and placing the 96-well plate Incubate on a shaker at room temperature for 1.5 hours at a shaking state at a shaking speed of 100 rpm. After the incubation time expires, the liquid in each sample well is exhausted, and the method is cleaned as described in step (3). 96-well plates.

(6) Add 100 μ 3′,3′,5,5′-tetradecylbenzidine to each well of the 96-well plate, and then incubate at room temperature for 10 minutes in the dark, after the incubation time expires. To each of the wells, 100 stop solution was added, and the stop solution was an aqueous solution prepared by sulfuric acid and sodium sulfite. The concentration of sulfuric acid was 2 mol/L, and the concentration of sodium citrate was 0.1 mol/L. (7) The 96-well plate is placed in a microplate reader, and the A450 value (optical density value or absorbance value) of each well is measured by an enzyme labeling instrument. The measured results are shown in the following table (the A450 value in the table is two holes). Weighted average).

Table 1: A450 values for standards

Table 2: A450 values of samples 1 ~ 20

Table 3: A450 values for samples 21 ~ 40

The A450 value of the blank control was 0.11045.

(8) According to the A450 value of each standard and the concentration of Adipsin in each standard, make A450 value -

The Adipsin concentration standard curve (see Figure 2), and get the formula y = 0.0008x + 0.0067, where y is the A450 value of the sample to be tested, and X is the content of Adipsin in the sample to be tested.

(9) Calculate the concentration of Adipsin in each sample according to the A450 value of each sample tested, using the formula y = 0.0008x + 0.0067. The calculation results are shown in the following table:

Table 4: Calculation results of samples 1 to 20 (Adipsin concentration unit: ng/mL) Sample 1 2 3 4 5 6 7

Adipsin 78.71875 39.03125 1105.094 434.4063 123.9375 908.5313 1423.125 Concentration

Sample 8 9 10 11 12 13 14

Adipsin 243.625 309.4063 79.71875 1613.656 50.8125 158.1875 195.5938 Concentration

Sample 15 16 17 18 19 20

Adipsin 23.1875 76.9063 146.9063 48.21875 91 77

Concentration Table 5: Calculation results of samples 21 to 40 (Adipsin concentration unit: ng/mL)

The average Adipsin concentration of samples 1-20 was calculated in Table 4, and the calculated result was 232.84±489.25 ng/mL. The average Adipsin concentration of sample 21 -40 in Table 5 was calculated and found to be 4.21 ± 3.14 ng/mL. From the calculation results, it can be seen that the difference between the average concentration of Adipsin in samples 1-20 from the eclampsia group and the average concentration of Adipsin in samples 21-40 from the healthy control group has statistical significance.

According to the calculation result of the mean ± 2χ standard deviation of the healthy control group, the detection limit is set. Therefore, the detection limit of the Adipsin set by the kit of the present invention is 12 ng/mL, that is, the concentration of Adipsin in the sample to be tested is higher than 12 ng/ The mL was judged to be positive (ie, with pre-eclampsia), and the Adipsin concentration was lower than 12 ng/mL (ie, did not have pre-eclampsia). The pre-eclampsia detection kit and the preparation method thereof using Adipsin as a detection index have been The present invention has been described with reference to the embodiments, and it will be apparent to those skilled in the art that the present invention can be modified or combined and modified as appropriate without departing from the spirit, scope, and scope of the present invention. . It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the spirit, scope and content of the invention.

Claims

Rights request

A pre-eclampsia detection kit using Adipsin as a detection index, characterized in that the kit comprises a multi-well plate coated with an anti-human Adipsin monoclonal antibody, and a biotin-labeled anti-human.

Adipsin monoclonal antibody detection solution, avidin-horseradish peroxidase bound to biotinylated anti-human Adipsin monoclonal antibody, chromogenic substrate 3', 3', 5, 5'-tetradecyl linkage Aniline and Adipsin protein standards.

2. The pre-eclampsia detection kit according to claim 1, wherein the perforated plate is coated with 0.9 to 1 g of anti-human Adipsin monoclonal antibody per well.

The pre-eclampsia detection kit using Adipsin as an indicator of detection according to claim 1 or 2, wherein the biotin-labeled anti-human Adipsin monoclonal antibody detection solution is mainly biotin-labeled anti-human Adipsin Monoclonal antibody, bovine serum albumin, glycerol and phosphate buffer, biotinylated anti-human Adipsin monoclonal antibody concentration of 24 mg / mL, bovine serum albumin concentration of 1.5 g ~ 3 g / 100 mL The concentration of glycerol is 50 mL/100 mL.

4. The pre-eclampsia detection kit according to claim 3, wherein the biotin-labeled anti-human Adipsin monoclonal antibody comprises biotinyl-N-hydroxysuccinimide ester. And the anti-human Adipsin monoclonal antibody is coupled, the mass ratio of the acyl-N-hydroxysuccinimide ester to the anti-human Adipsin monoclonal antibody is 0.5~1: 7.

The pre-eclampsia detection kit using Adipsin as an index of detection according to claim 1 or 2, further comprising a sample diluent, wherein the sample diluent is at a concentration of 0.01 mol/L to 0.02 mol/L. The phosphate buffer solution or the concentration of 0.01 mol/L ~ 0.02 mol/L of tris-hydroxydecylaminodecane buffer is prepared by adding bovine serum albumin. The concentration of bovine serum albumin is 1 g ~ 2 g / 100 mL. .

6. The pre-eclampsia detection kit according to claim 3, characterized in that the method further comprises a sample diluent, wherein the sample diluent is a phosphoric acid having a concentration of 0.01 mol/L to 0.02 mol/L. The concentration of bovine serum albumin is 1 g ~ 2 g / 100 mL in salt buffer or trihydroxydecylaminodecane buffer with a concentration of 0.01 mol/L ~ 0.02 mol/L.

7. The pre-eclampsia detection kit according to claim 4, further comprising a sample diluent, wherein the sample diluent is phosphoric acid at a concentration of 0.01 mol/L to 0.02 mol/L. The concentration of bovine serum albumin is lg ~ 2 g / 100 mL by adding buffer solution or a concentration of 0.01 mol/L to 0.02 mol/L of tris-hydroxydecylaminodecane buffer to bovine serum albumin.

8. A method for preparing a pre-eclampsia detection kit using Adipsin as a detection index, wherein the process steps are as follows:

The anti-human Adipsin monoclonal antibody was diluted to a concentration of 10 g/mL with a carbonate buffer of pH = 8-10, and anhydrous sterol was added to the anti-human Adipsin monoclonal antibody dilution. The amount of sterol added is limited to a concentration of 3 mL/100 mL, and then the sterol-containing anti-human Adipsin monoclonal antibody dilution is added to each well of the multiwell plate and coated at 4 ° C for at least 12 hours. After the coating time expires, the bovine serum albumin solution with a concentration of 1 g/100 mL is added to each well of the multi-well plate and blocked at 37 ° C for at least 1 hour; after the blocking reaction is completed, The porous plate was washed with a phosphate buffer having a pH of 7.2, and a porous plate coated with an anti-human Adipsin monoclonal antibody was obtained when the unreacted material on the porous plate was removed;

(2) Preparation of biotin-labeled anti-human Adipsin monoclonal antibody detection solution

1 using hydrazine, hydrazine-dihydrazinyl amide as solvent, biotinyl-hydrazine hydroxysuccinimide ester as solute to prepare a concentration of 50 g / L of acyl-N hydroxysuccinimide ester solution, to pH = 9.6 carbonate buffer is a solvent, anti-human Adipsin monoclonal antibody is a solute to prepare a concentration of 24 mg / mL of anti-human Adipsin monoclonal antibody solution, the acyl-N-hydroxysuccinimide ester solution and The mixture of the anti-human Adipsin monoclonal antibody solution is reacted at room temperature for at least 4 hours under stirring to obtain a biotin-labeled anti-human Adipsin monoclonal antibody;

After the reaction is completed, the reaction solution containing the biotin-labeled anti-human Adipsin monoclonal antibody is placed in a dialysis bag, and dialyzed at a pH of 9.2 phosphate buffer at 4 ° C for a dialysis time of at least 12 hours. The dialysate is added at least 3 times; and then the bovine serum albumin is added to the reaction solution after the dialysis, and the amount of the bovine serum albumin is limited to a concentration of 3 g to 6 g/100 mL;

2 The bio-labeled anti-human Adipsin monoclonal antibody prepared in step 1 and the dialysis reaction solution of bovine serum albumin and glycerol are measured at a volume ratio of 1:1, and uniformly mixed at room temperature to form biotin-labeled anti-human Adipsin. Monoclonal antibody detection solution;

(3) Equipped with avidin-horseradish peroxidase, chromogenic substrate 3', 3', 5,5'-tetradecylbenzidine and Adipsin protein standards.

9. The method for preparing a pre-eclampsia detection kit using Adipsin as a detection index according to claim 8, characterized in that a sample diluent is further provided, and the sample diluent is at a concentration of 0.01. Adding bovine serum albumin to a phosphate buffer solution of mol/L ~ 0.02 mol/L or a concentration of 0.01 mol/L to 0.02 mol/L of tris-hydroxydecylaminodecane buffer. The amount added is limited to a concentration of 1 g ~ 2 g / 100 mL.

The method for preparing a pre-eclampsia test kit using Adipsin as a detection index according to claim 8 or 9, wherein:

(1) When preparing a multi-well plate coated with an anti-human Adipsin monoclonal antibody, the anti-human Adipsin monoclonal antibody dilution is added in an amount of 100 μM / well, and the concentration is 1 g / 100 mL of bovine serum white The protein solution was added in an amount of 120 μΐ / hole;

(2) When the biotin-labeled anti-human Adipsin monoclonal antibody is prepared, the acyl-N is obtained by mass ratio of the acyl-N-hydroxysuccinimide ester to the anti-human Adipsin monoclonal antibody = 0.5 to 1:7. The hydroxysuccinimide ester solution and the anti-human Adipsin monoclonal antibody solution form a mixed solution.