CN104360086B - The latex enhancing immune of a kind of soluble leptin receptor is than turbid detection kit - Google Patents
The latex enhancing immune of a kind of soluble leptin receptor is than turbid detection kit Download PDFInfo
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Abstract
The present invention relates to external diagnosis reagent field, particularly relate to the latex enhancing immune of a kind of soluble leptin receptor than turbid detection kit. The present invention provides the latex enhancing immune of a kind of soluble leptin receptor than 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 by cross-linking the polystyrene latex microspheres of sOB-R antibody, buffer 2, stabilizer 2, preservative 2, protective agent 2 form, be wherein connected in covalent cross-linking mode between polystyrene latex microspheres with sOB-R antibody. The present invention adopts latex enhancing immune turbidimetry, when sOB-R and sOB-R antibody response in blood, has driven polystyrene latex to assemble and has produced certain turbidity, detected more convenient, it is easy to applied in clinic.
Description
Technical field
The present invention relates to external diagnosis reagent field, particularly relate to the latex enhancing immune of a kind of soluble leptin receptor than turbid detection kit.
Background technology
Soluble leptin receptor (solubleleptinreceptor, sOB-R) is topmost leptin-binding potein in blood circulation. Under native state, in blood of human body, leptin-binding potein has two kinds of sizes, molecular weight is 140kD and 110kD, after glycosidase desaccharide base, molecular weight is 90kD and 60kD, at human body, soluble leptin receptor comes off essentially from cross-film leptin receptor, and it has vital effect in regulating Leptin signaling function. SOB-R exists only in Normal human peripheral's blood circulation, not yet finds sOB-R in cerebrospinal fluid. Under physiological status, sOB-R is respectively provided with obvious circadian rhythm, and time hungry, sOB-R level raises. Healthy human body sOB-R is proportionate with high density lipoprotein (HDL) and adiponectin (adiponectin) level, with Body Mass Index (BMI), leptin level, fasting insulin and insulin resistance index (HOMA-IR) in negative correlation. In overweight people's body, sOB-R level reduces, and after Low calorie diet is lost weight, sOB-R level raises, and in the slimmer implementing stomach Partial Resection, sOB-R level raises. In Metabolic Syndrome Patients, sOB-R and insulin resistance index (HOMA-IR) are in negative correlation, and polycystic ovary syndrome patient, sOB-R level reduces. Clinical research finds, in serious Patients With Nephrotic Symdrome blood, sOB-R level raises, thus it is speculated that this reduction is probably the biochemical indicia (marker) of leptin resistance, is one of the constituent of metabolism syndrome.
Increasing clinical research displays that, under different physiology and pathologic condition, in the patient such as type 1 diabetes, obesity, the level of blood plasma sOB-R is different. Therefore, sOB-R is possibly through the semiotic function strengthening or weakening leptin, thus playing important adjustment effect in disease develops. The 916 example healthy young men of 18 years old has been investigated in a recent perspective study, and again investigated wherein 91 example experimenter after two years, find that the level of sOB-R becomes negative correlation with fat all basal measurements and metabolism risks and assumptions (blood pressure, total low-density lipoprotein cholesterol, fasting glucose), and with being proportionate property of HDL-C, this is the clinical research of first prospective larger samples so far, these results are pointed out, and sOB-R is likely to become Metabolic syndrome and seeks peace the warning index of fasting glucose.Research also finds that the level of blood plasma sOB-R is notable negative correlation with the risk of type 2 diabetes mellitus, after correcting the factors such as BMI, life style, diet, find that the risk of plasma leptin levels and type 2 diabetes mellitus does not have notable relatedness, but the risk of the level of sOB-R and type 2 diabetes mellitus is still in notable negative correlation. This relatedness is independent of BMI, leptin and adiponectin, and prompting sOB-R is the negativity index of type 2 diabetes mellitus risk independence. SOB-R detection can be widely used for endocrine, Gastroenterology dept., Infectious Disease, oncology, newborn intensive care unit, organ transplantation section and Experiment on therapy room etc.
SOB-R is detected, existing detection method mainly has quantitative detecting method, detection method has double-antibody method (ELISA), radio immunoassay (RIA) etc., wherein radio immunoassay has certain restriction in clinic, and current commonly used ELISA method detects. Although the sensitivity of ELISA is higher, but its complicated operation, it is necessary to more time, General Result is qualitative or sxemiquantitative, and time quantitative, deviation is relatively big, and the range of linearity is narrow, for the bad control of sample dilution.
Summary of the invention
For prior art above shortcomings, the technical problem to be solved is: provide a kind of preparation cost cheap, good stability, it is prone to preserve, data redundancy is good, and detection sensitivity is high, can be widely applied to the sOB-R detection kit of clinical biochemical instrument.
For achieving the above object and other relevant purposes, first aspect present invention provides the latex enhancing immune of a kind of soluble leptin receptor than turbid detection kit, it is characterized in that, it 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 by cross-linking the polystyrene latex microspheres of sOB-R antibody, buffer 2, stabilizer 2, preservative 2, protective agent 2 form, be wherein connected in covalent cross-linking mode between polystyrene latex microspheres with sOB-R antibody.
Preservative 1 and 2 in the technical program refers to and can suppress the class reagent that in reagent, antibacterial and microorganism are polluted, and reagent has the effect of antisepsis and sterilization. Protective agent one class in reagent R2 can protect the reagent of the antibody on latex particle surface. Stabilizer 1 and 2 can keep the charge balance in reagent. SOB-R latex enhancing immune in the technical program is than turbid test kit, by antibody linked for sOB-R on polystyrene latex microspheres surface, when sOB-R and sOB-R antibody response in blood, drive polystyrene latex microspheres to assemble and produce certain turbidity, and turbidity is directly proportional in certain limit to the sOB-R content in blood, it is possible to detect the content of sOB-R in blood under 400-800nm wavelength with full automatic biochemical apparatus.
Preferably, one or more the combination in Hepes buffer, Tris-HCl buffer, MOPS buffer, PBS, glycine buffer, borate buffer solution of the buffer 1 in described reagent R1, its pH is 7.0～9.0, and concentration is 25～500mmol/L; 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.
Buffer 1 in reagent R1 of the present invention can use the various conventional buffer in above-mentioned this area, but so that test kit has sensitivity more preferably and color developing effect, in heretofore described reagent R1, buffer 1 preferably comprises following component: the aqueous solution of stachyose, Alumen, Fructose Diphosphate sodium, sodium hexameta phosphate and glycine, and the total concentration of stachyose, Alumen, Fructose Diphosphate sodium, sodium hexameta phosphate, glycine is 3.5-7.5g/L, the pH value of buffer is 7.2-7.6.
Preferably, each component concentration in buffer 1 is:
The solvent of described buffer 1 is water.
Described buffer 1 can use the various conventional pH adjusting agent in this area to carry out the adjustment of pH value.
Preferably, one or more the combination in KCl, NaCl, CaCl of the stabilizer 1 in described reagent R1, its mass concentration is 0.5%～10%; Stabilizer 2 in described reagent R2 adopt ion stabilizer and suspension stabilizer with the use of; Wherein ion stabilizer is NaCl, KCl, Na2CO3、Na2SO4 or K2SO4, suspension stabilizer is PEG8000, sucrose, glycerol or glucose.
Stabilizer 1 one or more the combination in KCl, NaCl, CaCl in technique scheme, its mass concentration is 0.5%～10%, and such stabilizer 1 has the advantage that cheap, raw material is easy to get. Stabilizer 2 adopt ion stabilizer and suspension stabilizer with the use of; Wherein ion stabilizer is NaCl, KCl, Na2CO3、Na2SO4 or K2SO4, suspension stabilizer is PEG8000, sucrose, glycerol or glucose; Wherein preferably NaCl and sucrose with the use of, system so can be kept steady in a long-term.
Preferably, 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. Such preservative 1 and 2 has excellent antisepsis and sterilization performance.
Preferably, the solidifying agent of the increasing in described reagent R1 is PEG8000 or glucosan.
It is furthermore preferred that the solidifying agent of increasing in described reagent R1 is PEG8000, being owing to PEG8000 belongs to non-ionic water-soluble polymer, the dissolubility in water is relatively larger, it is possible to regulate the viscosity of reagent R1, promotes that antigen and antibody molecule are combined into complex.
Preferably, the surface functional group of the polystyrene latex microsphere in described reagent R2 is amino, carboxyl, hydrazides, aldehyde radical or epoxy radicals, polystyrene latex microsphere particle diameter at 100～600nm.
It is furthermore preferred that described polystyrene latex microsphere is surface functional group is the polystyrene latex microspheres of carboxyl. This is owing to the functional group that polystyrene latex microspheres surface is carboxyl is easy to be activated by EDC, thus quick and sOB-R antibodies, increases coupling effect, it is ensured that the stability of test result and accuracy.
Preferably, the sOB-R antibody in described reagent R2 is one or more combination of mouse-anti people's sOB-R antibody, Goat anti human's sOB-RIgG antibody or the anti-human sOB-RIgG antibody of rabbit.
Preferably, the protective agent 1 in described reagent R1 is bovine serum albumin; Protective agent 2 in described reagent R2 is bovine serum albumin. Protective agent 1 and 2 in the technical program can protect the activity of the sOB-R antibody on polystyrene latex particles surface.
Preferably, the concentration of described EDTA is 10～100mmol/L.
Second aspect present invention provides the latex enhancing immune of described soluble leptin receptor than the preparation method of turbid detection kit, comprises 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: sOB-R antibody is carried out 4 DEG C of dialysis, then with buffer, sOB-R antibody is diluted to 2mg/ml, obtain sOB-R 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 sOB-R 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, stabilizer, protective agent is uniformly mixed and obtains reagent R2.
Third aspect present invention provides the latex enhancing immune of described soluble leptin receptor than the turbid detection kit purposes in sOB-R content detection field.
Compared with existing detection technique, there is advantages that
1. adopt latex enhancing immune turbidimetry, when sOB-R and sOB-R antibody response in blood, drive polystyrene latex to assemble and produce certain turbidity, and turbidity is directly proportional within the specific limits to the sOB-R content in blood, can detect under the wavelength of 400～800nm, detect more convenient, it is easy to apply in clinic.
2. the surface functional group of polystyrene latex microspheres is amino, carboxyl, hydrazides or epoxy radicals etc., the functional group on its surface can combine with the amino of antibody surface etc. and form covalent coupling structure, sOB-R antibody is made firmly to be combined in latex microsphere surface, ensure that the stability of R2, extend reagent effect duration.
3. the bulky grain polystyrene latex particles adopting particle diameter to be 100～600nm, has bigger particle diameter, adds turbidity during sOB-R and sOB-R antibody response in blood, thus increasing the sensitivity of test reaction, shortening the response time.
4. reagent R2 adopts ion stabilizer and suspension stabilizer to combine to use, makes the charge balance state of reagent, improve the sOB-R latex enhancing immune stability than turbid test kit so that sOB-R latex enhancing immune than the stability of turbid test kit up to 18 months.
Detailed description of the invention
Below by way of specific instantiation, embodiments of the present invention being described, those skilled in the art the content disclosed by this specification can understand other advantages and effect of the present invention easily. The present invention can also be carried out by additionally different detailed description of the invention or apply, and the every details in this specification based on different viewpoints and application, can also carry out various modification or change under the spirit without departing from the present invention.
Before further describing the specific embodiment of the invention, it should be appreciated that protection scope of the present invention is not limited to following specific specific embodiments; It is also understood that the term used in the embodiment of the present invention is to describe specific specific embodiments, rather than in order to limit the scope of the invention; In specification and claims of the present invention, unless additionally explicitly pointed out in literary composition, singulative " ", " one " and " this " include plural form.
When embodiment provides numerical range, it should be appreciated that unless the present invention is otherwise noted, between two end points and two end points of each numerical range, any one numerical value all can be selected for. Unless otherwise defined, the same meaning that all technology used in the present invention and scientific terminology and those skilled in the art of the present technique are generally understood that. Except the concrete grammar used in embodiment, equipment, material, record according to those skilled in the art's grasp to prior art and the present invention, it is also possible to use similar with the method described in the embodiment of the present invention, equipment, material or that be equal to any method of prior art, equipment and material to realize the present invention.
Unless otherwise indicated, the experimental technique that disclosed in this invention, detection method, preparation method all adopt the conventional molecular biology of the art, biochemistry, chromatin Structure and analysis, analytical chemistry, cell are cultivated, the routine techniques of recombinant DNA technology and association area. These technology are existing in existing document improves explanation, specifically can referring to the MOLECULARCLONING:ALABORATORYMANUAL such as Sambrook, Secondedition, ColdSpringHarborLaboratoryPress, 1989andThirdedition, 2001;Ausubel etc., CURRENTPROTOCOLSINMOLECULARBIOLOGY, john wiley & sons, NewYork, 1987andperiodicupdates; TheseriesMETHODSINENZYMOLOGY, AcademicPress, SanDiego; Wolffe, CHROMATINSTRUCTUREANDFUNCTION, Thirdedition, AcademicPress, SanDiego, 1998; METHODSINENZYMOLOGY, Vol.304, Chromatin (P.M.WassarmanandA.P.Wolffe, eds.), AcademicPress, SanDiego, 1999; And METHODSINMOLECULARBIOLOGY, Vol.119, ChromatinProtocols (P.B.Becker, ed.) HumanaPress, Totowa, 1999 etc.
Embodiment 1
The preparation of R1 reagent:
Weigh 1.9g stachyose, 0.5g Alumen, 1.9g Fructose Diphosphate sodium, 0.3g sodium hexameta phosphate, 1.8g glycine, 20gNaCl, 50gPEG8000,0.5g sodium azide, 25g bovine serum albumin and 14.2gEDTA and be dissolved in 0.8L distilled water, regulate pH to 7.4, be settled to 1L and namely obtain reagent R1.
The preparation of R2 reagent:
Mouse-anti people's sOB-R antibody adds pH be 7.4, concentration be that the PBS of 100mmol/L carries out 4 DEG C of dialysis, centre completes dialysis after changing water 3 times, adopt the PBS of 100mmol/L that mouse-anti people's sOB-R antibody is diluted to mouse-anti people's sOB-R antibody diluent that concentration is 2mg/ml; By the polystyrene latex microspheres distilled water wash that surface functional group is carboxyl.
Again with pH be 7.4, concentration be that above-mentioned washed polystyrene latex microspheres is diluted to mass concentration is 1% for the PBS of 100mmol/L. the EDC of 0.01g is added in the above-mentioned latex diluent of 1L, at room temperature stirring reaction 30min, reaction is centrifuged after terminating and washs away unreacted EDC with PBS, it is subsequently adding step mouse-anti obtained above people's sOB-R antibody diluent, under room temperature after stirring reaction 1h, add 50g bovine serum albumin and terminate reaction, the reactant liquor obtained is used distilled water wash 3 times under the rotating speed of 15000rpm, add buffer 2 (25mM glycine buffer pH7.4), 50g bovine serum albumin, 0.5g sodium azide, 40gNaCl and 75g sucrose, it is settled to 1L, it is uniformly mixed and obtains reagent R2.
Reagent Beckman AU480 full automatic biochemical apparatus prepared by embodiment is tested, test wavelength is 570nm, commplementary wave length 800nm, sample this or calibration object 20ul, add the reagent R1 of 150ul, 37 DEG C of constant temperature 5min, it is subsequently adding reagent R2, read absorbance A 1,37 DEG C after 20s to hatch 4 points and read absorbance A 2 after 40 seconds, then reflection absorbance △ A=A2-A1; Carry out multiple spot calibration first by standard substance, and be calculated by spline function, obtain calibration curve. Sample is changed by absorbance, carries out contrast with standard curve and can obtain sOB-R concentration in sample. Above-described embodiment has carried out the checkings such as sensitivity for analysis, accuracy, elaboration and stability, and the result is as follows:
The sOB-R detection kit of embodiment 1 preparation carries out performance test, main its sensitivity for analysis of test, minimum detectability, accuracy, repeatability, stability and anti-interference etc.
1) lowest detectable limit: adopting 5%BSA normal saline solution as dummy, dummy should without measured object. Biochemistry analyzer continuously repeats detection 20 times, records testing result.Result shows that its lowest detection is limited to 0.1pg/mL.
2) accuracy: select the conventional sense sample of suitable concn, adds different amounts of numeraire product in conventional sample and is fabricated to reclaim sample, and definite value sample deionized water is as solvent; The deionized water adding same amount in conventional sample is fabricated to basis sample, and the amount of the numeraire product of addition is less than the 1/10 of cumulative volume, and 3 detections of each repetition take its average for reclaiming concentration. Result display average recovery rate is 100.1%, and accuracy is higher.
3) repeatability: do 2 batches of tests every day, every batch of serum sample taking same concentration is 2 mensuration, log, METHOD FOR CONTINUOUS DETERMINATION 20 days, result display CVIn batchIt is 1.35%, CVBetween batchIt is 2.14%, CVBetween itBeing 2.53%, total precision is 3.01%, and repeatability is better.
4) stability: take soB-R detection kit and carry out normal storage stability test, places for 2-8 DEG C and temporally within 2,4,6,8,9,10,11,12,13,14,15,16,17,18,19,20 months, detects respectively; Stability test of uncapping is measured by 2-8 DEG C of placement respectively for 0 day, 7 days, 14 days, 16 days, 18 days, 20 days, 22 days, 24 days, 26 days, 28 days, 30 days, 32 days. Result display soB-R detection kit is stored in 2-8 DEG C, in light protected environment, effect duration is 18 months. Uncap and be stored in 2-8 DEG C, in light protected environment, effect duration is 30 days.
Embodiment 2
The preparation of R1 reagent:
Weigh 1.9g stachyose, 0.5g Alumen, 1.9g Fructose Diphosphate sodium, 0.3g sodium hexameta phosphate, 1.8g glycine, 9gNaCl, 15gPEG8000,0.5g sodium azide, 5g bovine serum albumin and 14.5gEDTA and be dissolved in 0.8L distilled water, regulate pH to 7.4, be settled to 1L and namely obtain reagent R1.
The preparation of R2 reagent:
Mouse-anti people's sOB-R antibody adds pH be 7.4, concentration be that the PBS of 100mmol/L carries out 4 DEG C of dialysis, centre completes dialysis after changing water 3 times, adopt the PBS of 100mmol/L that mouse-anti people's sOB-R antibody is diluted to mouse-anti people's sOB-R antibody diluent that concentration is 2mg/ml; By the polystyrene latex microspheres distilled water wash that surface functional group is carboxyl.
Again with pH be 7.4, concentration be that above-mentioned washed polystyrene latex microspheres is diluted to mass concentration is 1% for the PBS of 100mmol/L. the EDC of 0.01g is added in the above-mentioned latex diluent of 1L, at room temperature stirring reaction 30min, reaction is centrifuged after terminating and washs away unreacted EDC with PBS, it is subsequently adding step mouse-anti obtained above people's sOB-R antibody diluent, under room temperature after stirring reaction 1h, add 50g bovine serum albumin and terminate reaction, the reactant liquor obtained is used distilled water wash 3 times under the rotating speed of 15000rpm, add buffer 2 (25mM glycine buffer pH7.4), 50g bovine serum albumin, 0.5g sodium azide, 9gNaCl and 80g sucrose, it is settled to 1L, it is uniformly mixed and obtains reagent R2.
Reagent Beckman AU480 full automatic biochemical apparatus prepared by embodiment is tested, test wavelength is 570nm, commplementary wave length 800nm, sample this or calibration object 20ul, add the reagent R1 of 150ul, 37 DEG C of constant temperature 5min, it is subsequently adding reagent R2, read absorbance A 1,37 DEG C after 20s to hatch 4 points and read absorbance A 2 after 40 seconds, then reflection absorbance △ A=A2-A1; Carry out multiple spot calibration first by standard substance, and be calculated by spline function, obtain calibration curve. Sample is changed by absorbance, carries out contrast with standard curve and can obtain sOB-R concentration in sample.Above-described embodiment has carried out the checkings such as sensitivity for analysis, accuracy, elaboration and stability, and the result is as follows:
Adopt the detection method identical with embodiment 1, verify that its detection is limited to 0.1pg/ml, accuracy (response rate) 99.80%, precision (CV) 3.05%, stability 18 months.
According to its test result it can be seen that detection kit provided by the present invention, there is good sensitivity for analysis, accuracy, elaboration and stability.
Embodiment 3
The preparation of R1 reagent:
Weigh 1.8g glycine, 9gNaCl, 15gPEG8000,0.5g sodium azide, 5g bovine serum albumin and 14.5gEDTA and be dissolved in 0.8L distilled water, regulate pH to 7.4, be settled to 1L and namely obtain reagent R1.
The preparation of R2 reagent:
Mouse-anti people's sOB-R antibody adds pH be 7.4, concentration be that the PBS of 100mmol/L carries out 4 DEG C of dialysis, centre completes dialysis after changing water 3 times, adopt the PBS of 100mmol/L that mouse-anti people's sOB-R antibody is diluted to mouse-anti people's sOB-R antibody diluent that concentration is 2mg/ml; By the polystyrene latex microspheres distilled water wash that surface functional group is carboxyl.
Again with pH be 7.4, concentration be that above-mentioned washed polystyrene latex microspheres is diluted to mass concentration is 1% for the PBS of 100mmol/L. the EDC of 0.01g is added in the above-mentioned latex diluent of 1L, at room temperature stirring reaction 30min, reaction is centrifuged after terminating and washs away unreacted EDC with PBS, it is subsequently adding step mouse-anti obtained above people's sOB-R antibody diluent, under room temperature after stirring reaction 1h, add 50g bovine serum albumin and terminate reaction, the reactant liquor obtained is used distilled water wash 3 times under the rotating speed of 15000rpm, add buffer 2 (25mM glycine buffer pH7.4), 50g bovine serum albumin, 0.5g sodium azide, 9gNaCl and 80g sucrose, it is settled to 1L, it is uniformly mixed and obtains reagent R2.
Reagent Beckman AU480 full automatic biochemical apparatus prepared by embodiment is tested, test wavelength is 570nm, commplementary wave length 800nm, sample this or calibration object 20ul, add the reagent R1 of 150ul, 37 DEG C of constant temperature 5min, it is subsequently adding reagent R2, read absorbance A 1,37 DEG C after 20s to hatch 4 points and read absorbance A 2 after 40 seconds, then reflection absorbance △ A=A2-A1; Carry out multiple spot calibration first by standard substance, and be calculated by spline function, obtain calibration curve. Sample is changed by absorbance, carries out contrast with standard curve and can obtain sOB-R concentration in sample. Above-described embodiment has carried out the checkings such as sensitivity for analysis, accuracy, elaboration and stability, and the result is as follows:
Adopt the detection method identical with embodiment 1, verify that its detection is limited to 3pg/ml, accuracy (response rate) 100.80%, precision (CV) 4.05%, stability 12 months.
In sum, the present invention effectively overcomes various shortcoming of the prior art and has high industrial utilization.
Above-described embodiment is illustrative principles of the invention and effect thereof only, not for the restriction present invention. Above-described embodiment all under the spirit and category of the present invention, can be modified or change by any those skilled in the art. Therefore, art has usually intellectual such as modifying without departing from all equivalences completed under disclosed spirit and technological thought or change, must be contained by the claim 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.
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