CN104360084B - A kind of ion stabilizer and suspension stabilizer with the use of sOB-R latex strengthen immunoturbidimetry detection kit - Google Patents

A kind of ion stabilizer and suspension stabilizer with the use of sOB-R latex strengthen immunoturbidimetry detection kit Download PDF

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CN104360084B
CN104360084B CN201410736436.7A CN201410736436A CN104360084B CN 104360084 B CN104360084 B CN 104360084B CN 201410736436 A CN201410736436 A CN 201410736436A CN 104360084 B CN104360084 B CN 104360084B
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吴勇泉
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Chongqing Zhongyuan Biotechnology Co Ltd
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials

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Abstract

The present invention relates to external diagnosis reagent field, particularly relate to a kind of ion stabilizer and suspension stabilizer with the use of sOB-R latex strengthen immunoturbidimetry detection kit.The invention provides a kind of ion stabilizer and suspension stabilizer with the use of sOB-R latex strengthen immunoturbidimetry detection kit, comprise reagent R1 and reagent R2, described reagent R1 by damping fluid 1, stabilizing agent 1, antiseptic 1, EDTA, agent is coagulated in increasing and protective agent 1 forms; Described reagent R2 by being cross-linked the polystyrene latex microspheres of sOB-R antibody, damping fluid 2, stabilizing agent 2, antiseptic 2, protective agent 2 form; wherein be connected in covalent cross-linking mode between polystyrene latex microspheres with sOB-R antibody, the stabilizing agent 2 in described reagent R2 adopt ion stabilizer and suspension stabilizer with the use of.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, easily in clinical middle application.

Description

A kind of ion stabilizer and suspension stabilizer with the use of sOB-R latex strengthen immunoturbidimetry detection kit
Technical field
The present invention relates to external diagnosis reagent field, particularly relate to a kind of ion stabilizer and suspension stabilizer with the use of sOB-R latex strengthen immunoturbidimetry 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 desugar base, molecular weight is 90kD and 60kD, at human body, soluble leptin receptor coming off mainly from cross-film leptin receptor, it has vital effect in adjustment Leptin signaling function.SOB-R is only present in Normal human peripheral's blood circulation, not yet finds sOB-R in cerebrospinal fluid.Under physiological status, sOB-R all has obvious circadian rhythm, and time hungry, sOB-R level raises.Healthy human body sOB-R and high-density lipoprotein (HDL) (HDL) and adiponectin (adiponectin) level are proportionate, with body mass index (BMI), leptin level, FPI and insulin resistance index (HOMA-IR) in negative correlation.In overweight people's body, sOB-R level reduces, and after Low calorie diet fat-reducing, 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, and inferring that this reduction may be the biochemical indicia (marker) of leptin resistance, is one of constituent of metabolic syndrome.
Increasing clinical research also shows, and under different physiology and pathologic condition, as in the patient such as type 1 diabetes, obesity, the level of blood plasma sOB-R is different.Therefore, sOB-R can by strengthening or weaken the semiotic function of leptin, thus play important regulating action in the developing of disease.The 916 example healthy young men of 18 years old has been investigated in recent perspective study, and again investigated wherein 91 routine experimenters after two years, find that the level of sOB-R becomes negative correlation with fat all basal measurements with metabolic risks and assumptions (blood pressure, total LDL-C, fasting blood-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 may become Metabolic syndrome and to seek peace the warning index of fasting blood-glucose.Research also finds that the level of blood plasma sOB-R and the risk of diabetes B are remarkable negative correlation, after correcting the factors such as BMI, life style, diet, find that the risk of plasma leptin levels and diabetes B does not have remarkable relevance, but the risk of the level of sOB-R and diabetes B is still in remarkable negative correlation.This relevance does not rely on BMI, leptin and adiponectin, and prompting sOB-R is diabetes B risk independently negativity index.SOB-R detects can be widely used in endocrine, Gastroenterology dept., Infectious Disease, oncology, newborn intensive care unit, organ transplant section and Experiment on therapy room etc.
SOB-R is detected, existing detection method mainly contains quantitative detecting method, detection method has double-antibody method (ELISA), radio immunoassay (RIA) etc., wherein radio immunoassay has certain restriction in clinical, and application ELISA method detects usually at present.Although the sensitivity of ELISA is higher, its complicated operation, needs the more time, and General Result is qualitative or sxemiquantitative, and time quantitative, deviation is comparatively large, and the range of linearity is narrow, for the bad control of sample dilution.
Summary of the invention
For prior art above shortcomings, technical matters to be solved by this invention is: provide a kind of preparation cost cheap, good stability, be easy to preserve, data redundancy is good, and detection sensitivity is high, can be widely used in the sOB-R detection kit of clinical biochemical instrument.
For achieving the above object and other relevant objects, first aspect present invention provide a kind of ion stabilizer and suspension stabilizer with the use of sOB-R latex strengthen immunoturbidimetry detection kit, it is characterized in that, comprise reagent R1 and reagent R2, described reagent R1 by damping fluid 1, stabilizing agent 1, antiseptic 1, EDTA, agent is coagulated in increasing and protective agent 1 forms; Described reagent R2 by being cross-linked the polystyrene latex microspheres of sOB-R antibody, damping fluid 2, stabilizing agent 2, antiseptic 2, protective agent 2 form, be wherein connected in covalent cross-linking mode between polystyrene latex microspheres with sOB-R antibody.
Antiseptic 1 and 2 in the technical program refers to a class reagent that can suppress bacterium and microbial contamination in reagent, reagent is had to the effect of antisepsis and sterilization.Protective agent one class in reagent R2 can protect the reagent of the antibody of latex particle surface.Stabilizing agent 1 and 2 can keep the charge balance in reagent.SOB-R latex enhancing immune in the technical program is than turbid 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 and the sOB-R content in blood are directly proportional in certain limit, the content of sOB-R in blood can be detected with full automatic biochemical apparatus under 400-800nm wavelength.
Preferably, damping fluid 1 in described reagent R1 is selected from one or more the combination in Hepes damping fluid, Tris-HCl damping fluid, MOPS damping fluid, PBS damping fluid, glycine buffer, borate buffer solution, its pH is 7.0 ~ 9.0, and concentration is 25 ~ 500mmol/L; Damping fluid 2 in described reagent R2 is selected from one or more in PBS damping fluid, borate buffer solution, glycine buffer, Hepes damping fluid, GOODS damping fluid, MOPS damping fluid, and its pH is 7.0 ~ 9.0, and concentration is 25 ~ 500mmol/L.
Damping fluid 1 in reagent R1 of the present invention can use the various conventional damping fluid in above-mentioned this area, but in order to make kit, there is better sensitivity and color developing effect, in the R1 of reagent described in the present invention, damping fluid 1 preferably comprises following component: the aqueous solution of stachyose, alum, Fructose Diphosphate, sodium hexametaphosphate and glycocoll, and the total concentration of stachyose, alum, Fructose Diphosphate, sodium hexametaphosphate, glycocoll is 3.5-7.5g/L, the pH value of damping fluid is 7.2-7.6.
Preferably, the concentration of each component in damping fluid 1 is:
The solvent of described damping fluid 1 is water.
Described damping fluid 1 can use the various conventional pH adjusting agent in this area to carry out the adjustment of pH value.
Preferably, the stabilizing agent 1 in described reagent R1 is selected from KCl, NaCl, CaCl 2in one or more combination, its mass concentration is 0.5% ~ 10%; Stabilizing agent 2 in described reagent R2 adopt ion stabilizer and suspension stabilizer with the use of; Wherein ion stabilizer is NaCl, KCl, Na 2cO3, Na 2sO4 or K 2sO4, suspension stabilizer is PEG8000, sucrose, glycerine or glucose.
In technique scheme, stabilizing agent 1 is selected from KCl, NaCl, CaCl 2in one or more combination, its mass concentration is 0.5% ~ 10%, and such stabilizing agent 1 has the advantage that cheap, raw material is easy to get.Stabilizing agent 2 adopt ion stabilizer and suspension stabilizer with the use of; Wherein ion stabilizer is NaCl, KCl, Na 2cO3, Na 2sO4 or K 2sO4, suspension stabilizer is PEG8000, sucrose, glycerine or glucose; Wherein preferably NaCl and sucrose with the use of, system can be kept so steady in a long-term.
Preferably, the antiseptic 1 in described reagent R1 is Sodium azide, thimerosal or ProClin300; Antiseptic 2 in described reagent R2 is Sodium azide, thimerosal or ProClin300.Such antiseptic 1 and 2 has excellent antisepsis and sterilization performance.
Preferably, it is PEG8000 or glucosan that agent is coagulated in the increasing in described reagent R1.
Preferred, it is PEG8000 that agent is coagulated in the increasing in described reagent R1, and be because PEG8000 belongs to non-ionic water-soluble polymer, the dissolubility in water is larger, can regulate the viscosity of reagent R1, promotes that antigen and antibody molecule are combined into compound.
Preferably, the surface functional group of the polystyrene latex microballoon in described reagent R2 is amino, carboxyl, hydrazides, aldehyde radical or epoxy radicals, polystyrene latex microballoon particle diameter at 100 ~ 600nm.
Preferred, the polystyrene latex microspheres of described polystyrene latex microballoon to be surface functional group be carboxyl.This is that the functional group being carboxyl due to polystyrene latex microspheres surface is easy to be activated by EDC, thus is combined with sOB-R antibody fast, increases coupling effect, ensures stability and the accuracy of test result.
Preferably, the sOB-R antibody in described reagent R2 is one or more combination of mouse-anti people sOB-R antibody, Goat anti human 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(BSA); Protective agent 2 in described reagent R2 is bovine serum albumin(BSA).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 described sOB-R latex to strengthen the preparation method of immunoturbidimetry detection kit, comprises the steps:
(1) preparation of reagent R1:
In damping fluid 1, add stabilizing agent 1, increase solidifying agent, antiseptic 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 damping fluid by sOB-R antibody dilution to 2mg/ml, obtain sOB-R antibody diluent; By centrifugal for polystyrene latex microspheres distilled water, washing 3 times;
Step 2: with damping fluid, 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% again, at room temperature stirring reaction 30min, reaction terminates rear 15000rpm centrifuge washing to remove unreacted EDC, then the sOB-R antibody diluent that step one obtains is added, stirred at ambient temperature reaction 30min, add stop buffer cessation reaction again, by centrifugal for the reactant liquor 15000rpm obtained, with damping fluid 2 washing precipitation, repeated centrifugation washs 3 times, finally add damping fluid 2, antiseptic, stabilizing agent, protective agent is uniformly mixed and obtains reagent R2.
Third aspect present invention provides described sOB-R latex to strengthen the purposes of immunoturbidimetry detection kit in sOB-R content detection field.
Compared with existing detection technique, the present invention has following beneficial effect:
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, easily in clinical middle application.
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 the reagent term of validity.
3. adopt particle diameter to be the bulky grain polystyrene latex particles of 100 ~ 600nm, there is larger particle diameter, add turbidity during sOB-R and sOB-R antibody response in blood, thus increase test reaction sensitivity, shorten the reaction time.
4. adopt ion stabilizer and suspension stabilizer to be combined in reagent R2, make the charge balance state of reagent, improve the stability of sOB-R latex enhancing immune than turbid kit, make sOB-R latex enhancing immune can reach 18 months than the stability of turbid kit.
Embodiment
Below by way of specific instantiation, embodiments of the present invention are described, those skilled in the art the content disclosed by this instructions can understand other advantages of the present invention and effect easily.The present invention can also be implemented or be applied by embodiments different in addition, and the every details in this instructions also can based on different viewpoints and application, carries out various modification or change not deviating under spirit of the present invention.
Before further describing the specific embodiment of the invention, should be understood 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, instead of in order to limit the scope of the invention; In instructions of the present invention and claims, unless explicitly pointed out in addition in literary composition, singulative " ", " one " and " this " comprise plural form.
When embodiment provides numerical range, should be understood that except non-invention is otherwise noted, between two end points of each numerical range and two end points, any one numerical value all can be selected.Unless otherwise defined, all technology used in the present invention are identical with the meaning that those skilled in the art of the present technique understand usually with scientific terminology.Except the concrete grammar used in embodiment, equipment, material, according to those skilled in the art to the grasp of prior art and record of the present invention, any method of prior art that is similar with the method described in the embodiment of the present invention, equipment, material or that be equal to, equipment and material can also be used to realize the present invention.
Unless otherwise indicated, disclosed in the present invention experimental technique, detection method, preparation method all adopt the routine techniques of the molecular biology of the art routine, biological chemistry, chromatin Structure and analysis, analytical chemistry, cell chulture, recombinant DNA technology and association area.These technology are existing in existing document improves explanation, specifically can see the MOLECULARCLONING:ALABORATORYMANUAL such as Sambrook, Secondedition, ColdSpringHarborLaboratoryPress, 1989andThirdedition, 2001; Ausubel etc., CURRENTPROTOCOLSINMOLECULARBIOLOGY, JohnWiley & 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:
Taking 1.9g stachyose, 0.5g alum, 1.9g Fructose Diphosphate, 0.3g sodium hexametaphosphate, 1.8g glycocoll, 20gNaCl, 50gPEG8000,0.5g Sodium azide, 25g bovine serum albumin(BSA) and 14.2gEDTA is 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:
In mouse-anti people sOB-R antibody, add the PBS damping fluid that pH is 7.4, concentration is 100mmol/L carry out 4 DEG C of dialysis, centre completes dialysis after changing water 3 times, adopts the PBS damping fluid of 100mmol/L to be the mouse-anti people sOB-R antibody diluent of 2mg/ml by mouse-anti people sOB-R antibody dilution to concentration; Be that the polystyrene latex microspheres distilled water of carboxyl washs by surface functional group.
Again with pH be 7.4, concentration is that above-mentioned washed polystyrene latex microspheres is diluted to mass concentration is 1% for the PBS damping fluid of 100mmol/L.The EDC of 0.01g is added in the above-mentioned latex dilution of 1L, at room temperature stirring reaction 30min, reaction terminates rear centrifugal and go out unreacted EDC with PBS buffer solution, then step mouse-anti people obtained above sOB-R antibody diluent is added, after stirred at ambient temperature reaction 1h, add 50g bovine serum albumin(BSA) cessation reaction again, the reactant liquor obtained is washed 3 times with distilled water under the rotating speed of 15000rpm, add damping fluid 2 (25mM glycine buffer pH7.4) again, 50g bovine serum albumin(BSA), 0.5g Sodium azide, 40gNaCl and 75g sucrose, be settled to 1L, be uniformly mixed and obtain 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, then add the reagent R1 of 150ul, 37 DEG C of constant temperature 5min, then reagent R2 is added, read absorbance A 1 after 20s, hatch 4 points for 37 DEG C and read absorbance A 2 afterwards in 40 seconds, then reflect absorbance △ A=A2-A1; First use standard items to carry out multiple spot calibration, and calculate by splines, obtain calibration curve.Sample is changed by absorbance, carries out contrast with typical curve and can obtain sOB-R concentration in sample.Above-described embodiment has been carried out to the checkings such as sensitivity for analysis, accuracy, accuracy and stability, the result is as follows:
SOB-R detection kit prepared by embodiment 1 carries out performance test, main its sensitivity for analysis of test, minimum detectability, accuracy, repeatability, stability and anti-interference etc.
1) lowest detectable limit: adopt 5%BSA normal saline solution as dummy, dummy should not contain measured object.Continuous duplicate detection 20 times on Biochemical Analyzer, record testing result.Result shows its lowest detection and is limited to 0.1pg/mL.
2) accuracy: the conventional sense sample selecting suitable concn, the numeraire product adding different amount in conventional sample are made into recovery sample, and definite value sample deionized water is as solvent; The deionized water adding same amount in conventional sample is made into basic sample, and the amount of the numeraire product added is no more than 1/10 of cumulative volume, and each repetition detects for 3 times gets 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, often criticize the serum sample getting same concentration and do 2 mensuration, log, METHOD FOR CONTINUOUS DETERMINATION 20 days, result display CV in batchbe 1.35%, CV between batchbe 2.14%, CV between itbe 2.53%, total precision is 3.01%, and repeatability better.
4) stability: get 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 measures 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, and the term of validity is 18 months.Uncap and be stored in 2-8 DEG C, in light protected environment, the term of validity is 30 days.
Embodiment 2
The preparation of R1 reagent:
Taking 1.9g stachyose, 0.5g alum, 1.9g Fructose Diphosphate, 0.3g sodium hexametaphosphate, 1.8g glycocoll, 9gNaCl, 15gPEG8000,0.5g Sodium azide, 5g bovine serum albumin(BSA) and 14.5gEDTA is 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:
In mouse-anti people sOB-R antibody, add the PBS damping fluid that pH is 7.4, concentration is 100mmol/L carry out 4 DEG C of dialysis, centre completes dialysis after changing water 3 times, adopts the PBS damping fluid of 100mmol/L to be the mouse-anti people sOB-R antibody diluent of 2mg/ml by mouse-anti people sOB-R antibody dilution to concentration; Be that the polystyrene latex microspheres distilled water of carboxyl washs by surface functional group.
Again with pH be 7.4, concentration is that above-mentioned washed polystyrene latex microspheres is diluted to mass concentration is 1% for the PBS damping fluid of 100mmol/L.The EDC of 0.01g is added in the above-mentioned latex dilution of 1L, at room temperature stirring reaction 30min, reaction terminates rear centrifugal and go out unreacted EDC with PBS buffer solution, then step mouse-anti people obtained above sOB-R antibody diluent is added, after stirred at ambient temperature reaction 1h, add 50g bovine serum albumin(BSA) cessation reaction again, the reactant liquor obtained is washed 3 times with distilled water under the rotating speed of 15000rpm, add damping fluid 2 (25mM glycine buffer pH7.4) again, 50g bovine serum albumin(BSA), 0.5g Sodium azide, 9gNaCl and 80g sucrose, be settled to 1L, be uniformly mixed and obtain 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, then add the reagent R1 of 150ul, 37 DEG C of constant temperature 5min, then reagent R2 is added, read absorbance A 1 after 20s, hatch 4 points for 37 DEG C and read absorbance A 2 afterwards in 40 seconds, then reflect absorbance △ A=A2-A1; First use standard items to carry out multiple spot calibration, and calculate by splines, obtain calibration curve.Sample is changed by absorbance, carries out contrast with typical curve and can obtain sOB-R concentration in sample.Above-described embodiment has been carried out to the checkings such as sensitivity for analysis, accuracy, accuracy and stability, the result is as follows:
Adopt the detection method identical with embodiment 1, verify that its detection is limited to 0.1pg/ml, accuracy (recovery) 99.80%, precision (CV) 3.05%, stability 18 months.
According to its test result, detection kit provided by the present invention, has good sensitivity for analysis, accuracy, accuracy and stability.
Embodiment 3
The preparation of R1 reagent:
Taking 1.8g glycocoll, 9gNaCl, 15gPEG8000,0.5g Sodium azide, 5g bovine serum albumin(BSA) and 14.5gEDTA is dissolved in 0.8L distilled water, regulates pH to 7.4, is settled to 1L and namely obtains reagent R1.
The preparation of R2 reagent:
In mouse-anti people sOB-R antibody, add the PBS damping fluid that pH is 7.4, concentration is 100mmol/L carry out 4 DEG C of dialysis, centre completes dialysis after changing water 3 times, adopts the PBS damping fluid of 100mmol/L to be the mouse-anti people sOB-R antibody diluent of 2mg/ml by mouse-anti people sOB-R antibody dilution to concentration; Be that the polystyrene latex microspheres distilled water of carboxyl washs by surface functional group.
Again with pH be 7.4, concentration is that above-mentioned washed polystyrene latex microspheres is diluted to mass concentration is 1% for the PBS damping fluid of 100mmol/L.The EDC of 0.01g is added in the above-mentioned latex dilution of 1L, at room temperature stirring reaction 30min, reaction terminates rear centrifugal and go out unreacted EDC with PBS buffer solution, then step mouse-anti people obtained above sOB-R antibody diluent is added, after stirred at ambient temperature reaction 1h, add 50g bovine serum albumin(BSA) cessation reaction again, the reactant liquor obtained is washed 3 times with distilled water under the rotating speed of 15000rpm, add damping fluid 2 (25mM glycine buffer pH7.4) again, 50g bovine serum albumin(BSA), 0.5g Sodium azide, 9gNaCl and 80g sucrose, be settled to 1L, be uniformly mixed and obtain 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, then add the reagent R1 of 150ul, 37 DEG C of constant temperature 5min, then reagent R2 is added, read absorbance A 1 after 20s, hatch 4 points for 37 DEG C and read absorbance A 2 afterwards in 40 seconds, then reflect absorbance △ A=A2-A1; First use standard items to carry out multiple spot calibration, and calculate by splines, obtain calibration curve.Sample is changed by absorbance, carries out contrast with typical curve and can obtain sOB-R concentration in sample.Above-described embodiment has been carried out to the checkings such as sensitivity for analysis, accuracy, accuracy and stability, the result is as follows:
Adopt the detection method identical with embodiment 1, verify that its detection is limited to 3pg/ml, accuracy (recovery) 100.80%, precision (CV) 4.05%, stability 12 months.
In sum, the present invention effectively overcomes various shortcoming of the prior art and tool high industrial utilization.
Above-described embodiment is illustrative principle of the present invention and effect thereof only, but not for limiting the present invention.Any person skilled in the art scholar all without prejudice under spirit of the present invention and category, can modify above-described embodiment or changes.Therefore, such as have in art usually know the knowledgeable do not depart from complete under disclosed spirit and technological thought all equivalence modify or change, must be contained by claim of the present invention.

Claims (12)

1. ion stabilizer and suspension stabilizer with the use of sOB-R latex strengthen an immunoturbidimetry detection kit, comprise reagent R1 and reagent R2, described reagent R1 by damping fluid 1, stabilizing agent 1, antiseptic 1, EDTA, agent is coagulated in increasing and protective agent 1 forms; Described reagent R2 by being cross-linked the polystyrene latex microballoon of sOB-R antibody, damping fluid 2, stabilizing agent 2, antiseptic 2, protective agent 2 form, wherein be connected in covalent cross-linking mode between polystyrene latex microballoon with sOB-R antibody, the stabilizing agent 2 in described reagent R2 adopt ion stabilizer and suspension stabilizer with the use of; Wherein ion stabilizer is NaCl, KCl, Na 2cO3, Na 2sO4 or K 2sO4, suspension stabilizer is PEG8000, sucrose, glycerine or glucose;
In described reagent R1, damping fluid 1 comprises following component: the aqueous solution of stachyose, alum, Fructose Diphosphate, sodium hexametaphosphate and glycocoll, and the total concentration of stachyose, alum, Fructose Diphosphate, sodium hexametaphosphate, glycocoll is 3.5-7.5g/L, the pH value of damping fluid is 7.2-7.6, and the concentration of each component in damping fluid 1 is:
The solvent of described damping fluid 1 is water.
2. detection kit as claimed in claim 1, it is characterized in that, the damping fluid 2 in described reagent R2 is selected from one or more in PBS damping fluid, borate buffer solution, GOODS damping fluid, and its pH is 7.0 ~ 9.0, and concentration is 25 ~ 500mmol/L.
3. detection kit as claimed in claim 2, it is characterized in that, described GOODS damping fluid is selected from one or more in glycine buffer, Hepes damping fluid, MOPS damping fluid.
4. detection kit as claimed in claim 1, it is characterized in that, the stabilizing agent 1 in described reagent R1 is selected from KCl, NaCl, CaCl 2in one or more combination, its mass concentration is 0.5% ~ 10%.
5. detection kit as claimed in claim 1, it is characterized in that, the antiseptic 1 in described reagent R1 is Sodium azide, thimerosal or ProClin300; Antiseptic 2 in described reagent R2 is Sodium azide, thimerosal or ProClin300.
6. detection kit as claimed in claim 1, it is characterized in that, it is PEG8000 or glucosan that agent is coagulated in the increasing in described reagent R1.
7. detection kit as claimed in claim 1, it is characterized in that, the surface functional group of the polystyrene latex microballoon in described reagent R2 is amino, carboxyl, hydrazides, aldehyde radical or epoxy radicals, and the particle diameter of polystyrene latex microballoon is at 100 ~ 600nm.
8. detection kit as claimed in claim 1, is characterized in that, the polystyrene latex microballoon of described polystyrene latex microballoon to be surface functional group be carboxyl.
9. detection kit as claimed in claim 1, it is characterized in that, the sOB-R antibody in described reagent R2 is one or more combination of mouse-anti people sOB-R antibody, Goat anti human sOB-RIgG antibody or the anti-human sOB-RIgG antibody of rabbit.
10. detection kit as claimed in claim 1, it is characterized in that, the protective agent 1 in described reagent R1 is bovine serum albumin(BSA); Protective agent 2 in described reagent R2 is bovine serum albumin(BSA).
11. detection kit as claimed in claim 1, it is characterized in that, described EDTA concentration is 10 ~ 100mmol/L.
The preparation method of the detection kit as described in 12. claims as arbitrary in claim 1-11, comprises the steps:
(1) preparation of reagent R1:
In damping fluid 1, add stabilizing agent 1, increase solidifying agent, antiseptic 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 damping fluid by sOB-R antibody dilution to 2mg/ml, obtain sOB-R antibody diluent; By centrifugal for polystyrene latex microballoon distilled water, washing 3 times;
Step 2: with damping fluid, the polystyrene latex microballoon after step one is washed being diluted to mass concentration is 1%, add the EDC that mass concentration is 0.01%-0.1% again, at room temperature stirring reaction 30min, reaction terminates rear 15000rpm centrifuge washing to remove unreacted EDC, then the sOB-R antibody diluent that step one obtains is added, stirred at ambient temperature reaction 30min, add stop buffer cessation reaction again, by centrifugal for the reactant liquor 15000rpm obtained, with damping fluid 2 washing precipitation, repeated centrifugation washs 3 times, finally add damping fluid 2, antiseptic 2, stabilizing agent 2, protective agent 2 is uniformly mixed and obtains reagent R2.
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