CN104360086B - The latex enhancing immune of a kind of soluble leptin receptor is than turbid detection kit - Google Patents

Abstract

The invention relates to the field of in vitro diagnostic reagents, in particular to a latex-enhanced immune turbidimetric detection kit for soluble leptin receptors. The invention provides a latex-enhanced immunoturbidimetric detection kit for soluble leptin receptors, comprising reagent R1 and reagent R2, wherein the reagent R1 consists of a buffer 1, a stabilizer 1, a preservative 1, EDTA, a coagulant and The protective agent 1 is composed of; the reagent R2 is composed of polystyrene latex microspheres of cross-linked sOB-R antibody, buffer 2, stabilizer 2, preservative 2, and protective agent 2, wherein polystyrene latex microspheres and sOB -R antibodies are linked by covalent cross-linking. The invention adopts the latex-enhanced immune turbidity method, when the sOB-R in the blood reacts with the sOB-R antibody, the polystyrene latex is aggregated to generate a certain turbidity, the detection is more convenient, and it is easy to be applied in clinic.

Description

A latex-enhanced immunoturbidimetric detection kit for soluble leptin receptor

technical field

The invention relates to the field of in vitro diagnostic reagents, in particular to a latex-enhanced immune turbidimetric detection kit for soluble leptin receptors.

Background technique

Soluble leptin receptor (sOB-R) is the most important leptin-binding protein in blood circulation. In the natural state, there are two sizes of leptin-binding protein in human blood, with molecular weights of 140kD and 110kD. After deglycosylation by glycosidase, the molecular weights are 90kD and 60kD. In the human body, soluble leptin receptors mainly come from transmembrane leptin Shedding of the receptor, which has a crucial role in regulating leptin signaling function. sOB-R only exists in the peripheral blood circulation of normal people, and sOB-R has not been found in the cerebrospinal fluid. Under physiological conditions, sOB-R has obvious circadian rhythm, and the level of sOB-R increases when hungry. In healthy people, sOB-R is positively correlated with high-density lipoprotein (HDL) and adiponectin (adiponectin) levels, and negatively correlated with body mass index (BMI), leptin levels, fasting insulin and insulin resistance index (HOMA-IR). The level of sOB-R in the body of obese people is decreased, and the level of sOB-R is increased after losing weight with a low-calorie diet. In patients with metabolic syndrome, sOB-R was inversely correlated with insulin resistance index (HOMA-IR), and in patients with polycystic ovary syndrome, sOB-R levels were decreased. Clinical studies have found that the level of sOB-R in the blood of patients with severe nephrotic syndrome increases, and it is speculated that this decrease may be a biochemical marker of leptin resistance and is one of the components of metabolic syndrome.

More and more clinical studies have also shown that in different physiological and pathological conditions, such as type 1 diabetes and obesity, the level of plasma sOB-R is different. Therefore, sOB-R may play an important regulatory role in the occurrence and development of diseases by enhancing or weakening the signaling function of leptin. In a recent prospective study of 916 healthy 18-year-old young men, 91 of whom were re-investigated two years later, sOB-R levels were found to be correlated with all baseline measures of obesity and metabolic Risk factors (blood pressure, total low-density lipoprotein cholesterol, fasting blood glucose) were negatively correlated, while positively correlated with high-density lipoprotein cholesterol. This is the first prospective clinical study with a larger sample so far, and these results It is suggested that sOB-R may be an early warning indicator of metabolic syndrome and fasting blood glucose. The study also found that the level of plasma sOB-R was significantly negatively correlated with the risk of type 2 diabetes. After adjusting BMI, lifestyle, diet and other factors, it was found that the level of plasma leptin was not significantly correlated with the risk of type 2 diabetes, but The level of sOB-R was still significantly negatively correlated with the risk of type 2 diabetes. This association was independent of BMI, leptin, and adiponectin levels, suggesting that sOB-R is an independent negative indicator of type 2 diabetes risk. sOB-R detection can be widely used in endocrinology, gastroenterology, infection, oncology, neonatal intensive care unit, organ transplantation and treatment laboratories, etc.

For the detection of sOB-R, the existing detection methods mainly include quantitative detection methods, such as double-antibody sandwich method (ELISA), radioimmunoassay (RIA), etc. Among them, radioimmunoassay has certain limitations in clinical practice. Usually ELISA method is used for detection. Although the sensitivity of ELISA is high, its operation is complicated and requires a lot of time. Generally, the results are qualitative or semi-quantitative. The quantitative deviation is relatively large, and the linear range is narrow. It is difficult to control the dilution of samples.

Contents of the invention

In view of the above-mentioned deficiencies in the prior art, the technical problem to be solved by the present invention is to provide a sOB with low preparation cost, good stability, easy storage, good data repeatability, high detection sensitivity, and can be widely used in clinical biochemical instruments. -R detection kit.

In order to achieve the above and other related purposes, the first aspect of the present invention provides a latex-enhanced immunoturbidimetric detection kit for soluble leptin receptors, which is characterized in that it includes reagent R1 and reagent R2, and the reagent R1 is composed of a buffer 1. Stabilizer 1, preservative 1, EDTA, coagulant and protective agent 1; the reagent R2 consists of polystyrene latex microspheres cross-linked sOB-R antibody, buffer 2, stabilizer 2, preservative 2. Protective agent 2, in which polystyrene latex microspheres and sOB-R antibodies are linked by covalent cross-linking.

Preservatives 1 and 2 in the technical solution refer to a class of reagents that can inhibit bacterial and microbial contamination in reagents, and have antiseptic and bactericidal effects on reagents. The protective agent in reagent R2 is a reagent that can protect the antibody on the surface of latex particles. Stabilizers 1 and 2 maintain charge balance within the reagent. The sOB-R latex-enhanced immune turbidimetric kit in this technical solution cross-links the sOB-R antibody on the surface of polystyrene latex microspheres, and when the sOB-R in the blood reacts with the sOB-R antibody, the polystyrene The aggregation of vinyl latex microspheres produces a certain turbidity, and the turbidity is directly proportional to the sOB-R content in the blood within a certain range, and the sOB-R content in the blood can be detected by an automatic biochemical analyzer at a wavelength of 400-800nm.

Preferably, the buffer 1 in the reagent R1 is selected from one or more combinations of Hepes buffer, Tris-HCl buffer, MOPS buffer, PBS buffer, glycine buffer, and borax buffer. The pH is 7.0-9.0, and the concentration is 25-500mmol/L; the buffer solution 2 in the reagent R2 is selected from PBS buffer solution, borax buffer solution, glycine buffer solution, Hepes buffer solution, GOODS buffer solution, and MOPS buffer solution. One or more, the pH is 7.0-9.0, and the concentration is 25-500mmol/L.

The buffer 1 in the reagent R1 of the present invention can use various buffers commonly used in the art above, but in order to make the kit have better sensitivity and color development effect, the buffer 1 in the reagent R1 of the present invention preferably includes The following components: an aqueous solution of stachyose, alum, sodium fructose diphosphate, sodium hexametaphosphate, and glycine, and the total concentration of stachyose, alum, sodium fructose diphosphate, sodium hexametaphosphate, and glycine is 3.5-7.5 g /L, the pH of the buffer is 7.2-7.6.

Preferably, the concentration of each component in buffer 1 is:

The solvent of the buffer solution 1 is water.

The pH value of the buffer solution 1 can be adjusted using various commonly used pH regulators in the art.

Preferably, the stabilizer 1 in the reagent R1 is selected from one or more combinations of KCl, NaCl, and CaCl, and its mass concentration is 0.5% to 10%; the stabilizer 2 in the reagent R2 uses ion The stabilizer and suspension stabilizer are used together; the ion stabilizer is NaCl, KCl, Na ₂ CO3, Na ₂ SO4 or K ₂ SO4, and the suspension stabilizer is PEG8000, sucrose, glycerol or glucose.

In the above technical solution, the stabilizer 1 is selected from one or more combinations of KCl, NaCl, and CaCl, and its mass concentration is 0.5% to 10%. Such a stabilizer 1 has the advantages of low price and readily available raw materials. Stabilizer 2 is used in combination with ion stabilizers and suspension stabilizers; the ion stabilizers are NaCl, KCl, Na ₂ CO3, Na ₂ SO4 or K ₂ SO4, and the suspension stabilizers are PEG8000, sucrose, glycerin or glucose; NaCl is preferred Used in conjunction with sucrose, this can keep the system stable for a long time.

Preferably, the preservative 1 in the reagent R1 is sodium azide, thimerosal or ProClin300; the preservative 2 in the reagent R2 is sodium azide, thimerosal or ProClin300. Such preservatives 1 and 2 have excellent antiseptic and bactericidal properties.

Preferably, the coagulant in the reagent R1 is PEG8000 or dextran.

More preferably, the coagulant in the reagent R1 is PEG8000, because PEG8000 belongs to a non-ionic water-soluble polymer and has relatively large solubility in water, which can adjust the viscosity of the reagent R1 and promote the combination of antigen and antibody molecules as Complex.

Preferably, the surface functional groups of the polystyrene latex microspheres in the reagent R2 are amino groups, carboxyl groups, hydrazides, aldehyde groups or epoxy groups, and the particle diameter of the polystyrene latex microspheres is 100-600 nm.

More preferably, the polystyrene latex microspheres are polystyrene latex microspheres whose surface functional groups are carboxyl groups. This is because the carboxyl functional groups on the surface of the polystyrene latex microspheres are easily activated by EDC, thereby quickly combining with the sOB-R antibody, increasing the coupling effect, and ensuring the stability and accuracy of the test results.

Preferably, the sOB-R antibody in the reagent R2 is one or a combination of mouse anti-human sOB-R antibody, goat anti-human sOB-RIgG antibody or rabbit anti-human sOB-RIgG antibody.

Preferably, the protective agent 1 in the reagent R1 is bovine serum albumin; the protective agent 2 in the reagent R2 is bovine serum albumin. Protective agents 1 and 2 in this technical solution can protect the activity of the sOB-R antibody on the surface of polystyrene latex particles.

Preferably, the concentration of the EDTA is 10-100mmol/L.

The second aspect of the present invention provides a preparation method of the latex-enhanced immune turbidimetric detection kit for soluble leptin receptors, comprising the following steps:

(1) Preparation of reagent R1:

Add stabilizer 1, coagulant, preservative 1, protective agent 1 and EDTA into buffer 1, stir and mix evenly to obtain reagent R1;

(2) Preparation of reagent R2:

Step 1: Dialyze the sOB-R antibody at 4°C, then dilute the sOB-R antibody to 2 mg/ml with buffer to obtain the sOB-R antibody dilution; centrifuge and wash the polystyrene latex microspheres with distilled water 3 times ;

Step 2: Dilute the polystyrene latex microspheres washed in step 1 with buffer solution to a mass concentration of 1%, then add EDC with a mass concentration of 0.01%-0.1%, and stir and react at room temperature for 30 minutes. Centrifuge and wash at 15000rpm to remove unreacted EDC, then add the sOB-R antibody dilution obtained in step 1, stir and react at room temperature for 30min, then add stop solution to terminate the reaction, centrifuge the obtained reaction solution at 15000rpm, and wash the precipitate with buffer 2 , repeated centrifugation and washing 3 times, and finally added buffer 2, preservative, stabilizer, and protective agent, stirred and mixed evenly to obtain reagent R2.

The third aspect of the present invention provides the use of the soluble leptin receptor latex-enhanced immunoturbidimetric detection kit in the field of sOB-R content detection.

Compared with the existing detection technology, the present invention has the following beneficial effects:

1. Using latex-enhanced immune turbidimetry, when the sOB-R in the blood reacts with the sOB-R antibody, it drives the aggregation of polystyrene latex to produce a certain turbidity, and the turbidity is related to the sOB-R content in the blood It is directly proportional within a certain range, and can be detected at a wavelength of 400-800 nm. The detection is more convenient and easy to be applied in clinic.

2. The functional groups on the surface of polystyrene latex microspheres are amino, carboxyl, hydrazide or epoxy, etc., and the functional groups on the surface can combine with amino groups on the surface of the antibody to form a covalent coupling structure, making the sOB-R antibody firm Combined with the surface of latex microspheres, it ensures the stability of R2 and prolongs the validity period of the reagent.

3. The large particle size of polystyrene latex particles with a particle size of 100-600nm is used, which has a larger particle size, which increases the turbidity of the sOB-R and sOB-R antibody reaction in the blood, thereby increasing the sensitivity of the test reaction. Reduced reaction time.

4. The ion stabilizer and suspension stabilizer are used in combination in reagent R2 to make the charge balance state of the reagent, improve the stability of the sOB-R latex enhanced immune turbidimetric kit, and make the sOB-R latex enhanced immune turbidimetric kit Stability of up to 18 months.

detailed description

Embodiments of the present invention are described below through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation modes, and various modifications or changes can be made to the details in this specification based on different viewpoints and applications without departing from the spirit of the present invention.

Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the examples of the present invention are to describe specific specific embodiments, It is not intended to limit the protection scope of the present invention; in the description and claims of the present invention, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" include plural forms.

When the examples give numerical ranges, it should be understood that, unless otherwise stated in the present invention, the two endpoints of each numerical range and any value between the two endpoints can be selected. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to those skilled in the art's grasp of the prior art and the description of the present invention, the methods, equipment, and materials described in the embodiments of the present invention can also be used Any methods, apparatus and materials of the prior art similar or equivalent to the practice of the present invention.

Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in the technical field conventional technology.这些技术在现有文献中已有完善说明，具体可参见Sambrook等MOLECULARCLONING：ALABORATORYMANUAL，Secondedition，ColdSpringHarborLaboratoryPress，1989andThirdedition，2001；Ausubel等，CURRENTPROTOCOLSINMOLECULARBIOLOGY，JohnWiley&Sons，NewYork，1987andperiodicupdates；theseriesMETHODSINENZYMOLOGY，AcademicPress，SanDiego；Wolffe，CHROMATINSTRUCTUREANDFUNCTION， Thirdedition, Academic Press, San Diego, 1998; METHODSINENZYMOLOGY, Vol. 304, Chromatin (P.M. Wassarman and A.P. Wolffe, eds.), Academic Press, San Diego, 1999; and METHODSINMOLECULARBIOLOGY, Vol. Totowa, 1999 et al.

Example 1

Preparation of R1 reagent:

Weigh 1.9g stachyose, 0.5g alum, 1.9g sodium fructose diphosphate, 0.3g sodium hexametaphosphate, 1.8g glycine, 20gNaCl, 50gPEG8000, 0.5g sodium azide, 25g bovine serum albumin and 14.2g EDTA dissolved in Adjust the pH to 7.4 in 0.8L of distilled water, and set the volume to 1L to obtain reagent R1.

Preparation of R2 reagent:

Add PBS buffer solution with a pH of 7.4 and a concentration of 100mmol/L to the mouse anti-human sOB-R antibody for dialysis at 4°C, and complete the dialysis after changing the water 3 times in the middle. -R antibody diluted to a concentration of 2mg/ml mouse anti-human sOB-R antibody diluent; the polystyrene latex microspheres whose surface functional groups are carboxyl groups were washed with distilled water.

The washed polystyrene latex microspheres were then diluted to a mass concentration of 1% with PBS buffer solution having a pH of 7.4 and a concentration of 100 mmol/L. Add 0.01g of EDC to 1L of the above latex dilution, stir and react at room temperature for 30min, centrifuge after the reaction and wash with PBS buffer to remove unreacted EDC, then add the mouse anti-human sOB-R antibody dilution obtained in the above steps After stirring and reacting at room temperature for 1 h, 50 g of bovine serum albumin was added to terminate the reaction, and the obtained reaction solution was washed 3 times with distilled water at a rotating speed of 15000 rpm, and then buffer 2 (25 mM glycine buffer pH 7.4), 50 g Bovine serum albumin, 0.5g sodium azide, 40g NaCl and 75g sucrose, make up to 1L, stir and mix evenly to obtain reagent R2.

The reagents prepared in the examples were tested with a Beckman AU480 automatic biochemical instrument. The test wavelength was 570nm and the secondary wavelength was 800nm. Take 20ul of samples or calibration products, then add 150ul of reagent R1, keep the temperature at 37°C for 5min, and then add reagent R2 , read the absorbance A1 after 20s, and read the absorbance A2 after incubation at 37°C for 4 minutes and 40 seconds, then reflect the absorbance △A=A2-A1; marked curve. The concentration of sOB-R in the sample can be obtained by comparing the sample with the standard curve through the change of absorbance. The above-mentioned embodiment has been verified such as analysis sensitivity, accuracy, precision and stability, and verification result is as follows:

The performance test of the sOB-R detection kit prepared in Example 1 was mainly carried out to test its analytical sensitivity, minimum detection limit, accuracy, repeatability, stability and anti-interference, etc.

1) Minimum detection limit: 5% BSA saline solution is used as a blank sample, and the blank sample should not contain the analyte. The detection was repeated 20 times continuously on the biochemical analyzer, and the detection results were recorded. The results showed that the lowest detection limit was 0.1pg/mL.

2) Accuracy: Select a routine test sample with an appropriate concentration, add different amounts of deionized standard products to the routine sample to make a recovered sample, and use deionized water as a solvent for the deionized sample; add the same amount of deionized water to the routine sample The water is made into a basic sample, and the amount of the fixed value standard substance added does not exceed 1/10 of the total volume, and the average value of each test repeated 3 times is the recovery concentration. The results showed that the average recovery rate was 100.1%, and the accuracy was high.

3) Repeatability: Do 2 batches of tests every day, take the same concentration of serum samples for 2 measurements in each batch, record the test results, and measure continuously for 20 days. The results show that the CV _{within the batch} is 1.35%, the CV _{between the batches} is 2.14%, and the CV _{The day interval} is 2.53%, the total precision is 3.01%, and the repeatability is good.

4) Stability: Take the soB-R detection kit for routine storage stability test, place it at 2-8°C according to time 2, 4, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, and 20 months for testing; open the lid stability test according to 2-8 ℃ for 0 days, 7 days, 14 days, 16 days, 18 days, 20 days, 22 days, 24 days, 26 days days, 28 days, 30 days, and 32 days were measured. The results show that the soB-R detection kit is stored at 2-8°C in a light-proof environment, and the validity period is 18 months. Open the lid and store at 2-8°C in a dark environment, and the validity period is 30 days.

Example 2

Preparation of R1 reagent:

Weigh 1.9g stachyose, 0.5g alum, 1.9g sodium fructose diphosphate, 0.3g sodium hexametaphosphate, 1.8g glycine, 9gNaCl, 15gPEG8000, 0.5g sodium azide, 5g bovine serum albumin and 14.5gEDTA dissolved in Adjust the pH to 7.4 in 0.8L of distilled water, and set the volume to 1L to obtain reagent R1.

Preparation of R2 reagent:

Add PBS buffer solution with a pH of 7.4 and a concentration of 100mmol/L to the mouse anti-human sOB-R antibody for dialysis at 4°C, and complete the dialysis after changing the water 3 times in the middle. -R antibody diluted to a concentration of 2mg/ml mouse anti-human sOB-R antibody diluent; the polystyrene latex microspheres whose surface functional groups are carboxyl groups were washed with distilled water.

The washed polystyrene latex microspheres were then diluted to a mass concentration of 1% with PBS buffer solution having a pH of 7.4 and a concentration of 100 mmol/L. Add 0.01g of EDC to 1L of the above latex dilution, stir and react at room temperature for 30min, centrifuge after the reaction and wash with PBS buffer to remove unreacted EDC, then add the mouse anti-human sOB-R antibody dilution obtained in the above steps After stirring and reacting at room temperature for 1 h, 50 g of bovine serum albumin was added to terminate the reaction, and the obtained reaction solution was washed 3 times with distilled water at a rotating speed of 15000 rpm, and then buffer 2 (25 mM glycine buffer pH 7.4), 50 g Bovine serum albumin, 0.5g sodium azide, 9g NaCl and 80g sucrose, make up to 1L, stir and mix evenly to obtain reagent R2.

The reagents prepared in the examples were tested with a Beckman AU480 automatic biochemical instrument. The test wavelength was 570nm and the secondary wavelength was 800nm. Take 20ul of samples or calibration products, then add 150ul of reagent R1, keep the temperature at 37°C for 5min, and then add reagent R2 , read the absorbance A1 after 20s, and read the absorbance A2 after incubation at 37°C for 4 minutes and 40 seconds, then reflect the absorbance △A=A2-A1; marked curve. The concentration of sOB-R in the sample can be obtained by comparing the sample with the standard curve through the change of absorbance. The above-mentioned embodiment has been verified such as analysis sensitivity, accuracy, precision and stability, and verification result is as follows:

Using the same detection method as in Example 1, it was verified that the detection limit was 0.1 pg/ml, the accuracy (recovery rate) was 99.80%, the precision (CV) was 3.05%, and the stability was 18 months.

According to the test results, it can be seen that the detection kit provided by the present invention has better analytical sensitivity, accuracy, precision and stability.

Example 3

Preparation of R1 reagent:

Weigh 1.8g glycine, 9gNaCl, 15gPEG8000, 0.5g sodium azide, 5g bovine serum albumin and 14.5g EDTA, dissolve in 0.8L distilled water, adjust the pH to 7.4, and dilute to 1L to obtain reagent R1.

Preparation of R2 reagent:

Add PBS buffer solution with a pH of 7.4 and a concentration of 100mmol/L to the mouse anti-human sOB-R antibody for dialysis at 4°C, and complete the dialysis after changing the water 3 times in the middle. -R antibody diluted to a concentration of 2mg/ml mouse anti-human sOB-R antibody diluent; the polystyrene latex microspheres whose surface functional groups are carboxyl groups were washed with distilled water.

The washed polystyrene latex microspheres were then diluted to a mass concentration of 1% with PBS buffer solution having a pH of 7.4 and a concentration of 100 mmol/L. Add 0.01g of EDC to 1L of the above latex dilution, stir and react at room temperature for 30min, centrifuge after the reaction and wash with PBS buffer to remove unreacted EDC, then add the mouse anti-human sOB-R antibody dilution obtained in the above steps After stirring and reacting at room temperature for 1 h, 50 g of bovine serum albumin was added to terminate the reaction, and the obtained reaction solution was washed 3 times with distilled water at a rotating speed of 15000 rpm, and then buffer 2 (25 mM glycine buffer pH 7.4), 50 g Bovine serum albumin, 0.5g sodium azide, 9g NaCl and 80g sucrose, make up to 1L, stir and mix evenly to obtain reagent R2.

The reagents prepared in the examples were tested with a Beckman AU480 automatic biochemical instrument. The test wavelength was 570nm and the secondary wavelength was 800nm. Take 20ul of samples or calibration products, then add 150ul of reagent R1, keep the temperature at 37°C for 5min, and then add reagent R2 , read the absorbance A1 after 20s, and read the absorbance A2 after incubation at 37°C for 4 minutes and 40 seconds, then reflect the absorbance △A=A2-A1; marked curve. The concentration of sOB-R in the sample can be obtained by comparing the sample with the standard curve through the change of absorbance. The above-mentioned embodiment has been verified such as analysis sensitivity, accuracy, precision and stability, and verification result is as follows:

Using the same detection method as in Example 1, it was verified that the detection limit was 3 pg/ml, the accuracy (recovery rate) was 100.80%, the precision (CV) was 4.05%, and the stability was 12 months.

To sum up, the present invention effectively overcomes various shortcomings in the prior art and has high industrial application value.

The above-mentioned embodiments only illustrate the principles and effects of the present invention, but are not intended to limit the present invention. Anyone skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and technical ideas disclosed in the present invention shall still be covered by the claims of the present invention.

Claims (13)

1. the latex enhancing immune of soluble leptin receptor is than a turbid detection kit, is made up of buffer 1, stabilizer 1, preservative 1, EDTA, the solidifying agent of increasing and protective agent 1 including reagent R1 and reagent R2, described reagent R1, described reagent R2 is by the polystyrene latex microspheres of cross-linked soluble leptin receptor antibody, buffer 2, stabilizer 2, preservative 2, protective agent 2 forms, wherein it is connected in covalent cross-linking mode between polystyrene latex microspheres with soluble leptin receptor antibody, in described reagent R1, buffer 1 is for including following component: stachyose, Alumen, Fructose Diphosphate sodium, the aqueous solution of sodium hexameta phosphate and glycine, and stachyose, Alumen, Fructose Diphosphate sodium, sodium hexameta phosphate, the total concentration of glycine is 3.5-7.5g/L, the pH value of buffer is 7.2-7.6, each component concentration in buffer 1 is:

The solvent of described buffer 1 is water; One or more in PBS, borate buffer solution, glycine buffer, Hepes buffer, GOODS buffer, MOPS buffer of buffer 2 in described reagent R2, its pH is 7.0～9.0, and concentration is 25～500mmol/L.

2. detection kit as claimed in claim 1, it is characterised in that the stabilizer 2 in described reagent R2 adopt ion stabilizer and suspension stabilizer with the use of.

3. detection kit as claimed in claim 2, it is characterised in that wherein ion stabilizer is NaCl, KCl, Na₂CO₃、Na₂SO₄Or K₂SO₄。

4. detection kit as claimed in claim 2, it is characterised in that wherein suspension stabilizer is PEG8000, sucrose, glycerol or glucose.

5. detection kit as claimed in claim 1, it is characterised in that the stabilizer 1 in described reagent R1 is selected from KCl, NaCl, CaCl₂In one or more combination, its mass concentration is 0.5%～10%.

6. detection kit as claimed in claim 1, it is characterised in that the preservative 1 in described reagent R1 is sodium azide, thimerosal or ProClin300; Preservative 2 in described reagent R2 is sodium azide, thimerosal or ProClin300.

7. detection kit as claimed in claim 1, it is characterised in that the solidifying agent of the increasing in described reagent R1 is PEG8000 or glucosan.

8. detection kit as claimed in claim 1, it is characterised in that the surface functional group of the polystyrene latex microspheres in described reagent R2 is amino, carboxyl, hydrazides, aldehyde radical or epoxy radicals.

9. detection kit as claimed in claim 1, it is characterised in that the particle diameter of polystyrene latex microspheres is 100～600nm.

10. detection kit as claimed in claim 1, it is characterized in that, the soluble leptin receptor antibody in described reagent R2 is one or more combination of mouse-anti human soluble leptin receptor antibody, Goat anti human's soluble leptin receptor IgG antibody or the anti-human soluble leptin receptor IgG antibody of rabbit.

11. detection kit as claimed in claim 1, it is characterised in that the protective agent 1 in described reagent R1 is bovine serum albumin; Protective agent 2 in described reagent R2 is bovine serum albumin.

12. detection kit as claimed in claim 1, it is characterised in that described EDTA concentration is 10～100mmol/L.

13. the preparation method of the detection kit as described in claim 1-12 any claim, comprise the steps:

(1) preparation of reagent R1:

Buffer 1 adds stabilizer 1, increases solidifying agent, preservative 1, protective agent 1 and EDTA, be uniformly mixed, obtain reagent R1;

(2) preparation of reagent R2:

Step one: soluble leptin receptor antibody is carried out 4 DEG C of dialysis, then with buffer, soluble leptin receptor antibody is diluted to 2mg/ml, obtain soluble leptin receptor antibody diluent; Polystyrene latex microspheres distilled water is centrifuged, washs 3 times;

Step 2: with buffer, the polystyrene latex microspheres after step one is washed being diluted to mass concentration is 1%, add the EDC that mass concentration is 0.01%-0.1%, at room temperature stirring reaction 30min, reaction terminates rear 15000rpm centrifuge washing to remove unreacted EDC, it is subsequently adding the soluble leptin receptor antibody diluent that step one obtains, stirring reaction 30min under room temperature, add stop buffer and terminate reaction, the reactant liquor 15000rpm obtained is centrifuged, by buffer 2 washing precipitation, repeated centrifugation is washed 3 times, it is eventually adding buffer 2, preservative 2, stabilizer 2, protective agent 2 is uniformly mixed and obtains reagent R2.