CN104459105B - A kind of latex enhancing immune detecting LP is than turbid kit and preparation method thereof - Google Patents
A kind of latex enhancing immune detecting LP is than turbid kit and preparation method thereof Download PDFInfo
- Publication number
- CN104459105B CN104459105B CN201410736366.5A CN201410736366A CN104459105B CN 104459105 B CN104459105 B CN 104459105B CN 201410736366 A CN201410736366 A CN 201410736366A CN 104459105 B CN104459105 B CN 104459105B
- Authority
- CN
- China
- Prior art keywords
- damping fluid
- reagent
- antibody
- polystyrene latex
- agent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/82—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a precipitate or turbidity
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Hematology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Urology & Nephrology (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Plasma & Fusion (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention relates to external diagnosis reagent field, particularly relate to a kind of latex enhancing immune detecting LP than turbid kit.The invention provides a kind of latex enhancing immune detecting leptin (Leptin) than turbid 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 LP 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 LP antibody, the surface functional group of the polystyrene latex microballoon in described reagent R2 is amino, carboxyl, hydrazides, aldehyde radical or epoxy radicals.The present invention adopts latex enhancing immune turbidimetry, when LP and LP antibody response in blood, has driven polystyrene latex to assemble and has produced certain turbidity, detected more convenient, easily in clinical middle application.
Description
Technical field
The present invention relates to external diagnosis reagent field, particularly relate to a kind of latex enhancing immune detecting LP than turbid kit.
Background technology
Leptin (Leptin) is a kind of molecular weight is 16kDa, the single chain protein of 146 amino acid compositions, and it is encoded by ob gene, the protein hormone of adipocyte synthesis secretion.People generally believe it to enter after blood circulation can involved in sugar, fat and energetic supersession adjustment, impel body to reduce and ingest, increase fault offset, the synthesis of suppression adipocyte, and then make to lose weight.Also relevant with inflammatory reaction, neuroendocrine, tumor in digestive tract, obstetrical and gynecological disease, liver diseases etc., the exception of its metabolism take part in the pathogenic process of relevant disease.The major function of leptin is adjusting energy balance, is the basic hormone maintaining normal type, and experiment proves that overweight people's Serum leptin levels is all higher than non-obese person.In malignant tumour, the expression of leptin and acceptor thereof and tumour, as closely related with prognosis in generations such as breast cancer, cancer of the stomach, liver cancer.In chronic liver disease, leptin level and severity extent significant correlation, no matter dysfunction of liver degree, its serum leptin level is all higher than normal person.In normal pregnancy, in blood, leptin level increases gradually with gestation progress, and during preeclampsia, leptin increases, therefore leptin can be used as a clinical indices.Research shows that premature infant weight decline and plasma leptin exist remarkable positive correlation between declining.Insulin stimulating leptin secretion under normal circumstances, meanwhile leptin is by suppressing the secretion of insulin with the receptors bind on islet cells, prompting leptin and insulin and Relationship between insulin resistance close.Research also finds, essential hypertension leptin and insulin level significantly raise, and become positive correlation each other.
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, is easy to preserve, and data redundancy is good, and detection sensitivity is high, can be widely used in the LP detection kit of clinical biochemical instrument.
For achieving the above object and other relevant objects, first aspect present invention provides a kind of latex enhancing immune detecting LP than turbid 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 LP 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 LP 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.LP latex enhancing immune in the technical program is than turbid kit, by antibody linked for LP on polystyrene latex microspheres surface, when LP and LP antibody response in blood, drive polystyrene latex microspheres to assemble and produce certain turbidity, and turbidity and the LP content in blood are directly proportional in certain limit, the content of LP 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 one or more the combination in KCl, NaCl, CaCl, and 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 one or more the combination in KCl, NaCl, CaCl, and 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 LP antibody fast, increases coupling effect, ensures stability and the accuracy of test result.
Preferably, the LP antibody in described reagent R2 is one or more combination of mouse-anti people LP antibody, Goat anti human LPIgG antibody or the anti-human LPIgG 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 LP 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 detection LP than the preparation method of turbid 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: LP antibody is carried out 4 DEG C of dialysis, then with damping fluid by LP antibody dilution to 2mg/ml, obtain LP 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 LP 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 the latex enhancing immune of described detection LP than the purposes of turbid kit in LP content detection field.
Compared with existing detection technique, the present invention has following beneficial effect:
1. adopt latex enhancing immune turbidimetry, when LP and LP antibody response in blood, drive polystyrene latex to assemble and produce certain turbidity, and turbidity is directly proportional within the specific limits to the LP 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, LP 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 LP and LP 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 LP latex enhancing immune than turbid kit, make LP 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 LP 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 LP antibody diluent of 2mg/ml by mouse-anti people LP 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 LP 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 LP 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:
Leptin 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 0.022ng/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 99.96%, 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 1.55%, CV
between batchbe 2.04%, CV
between itbe 2.63%, total precision is 3.11%, and repeatability better.
4) stability: get Leptin 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 Leptin 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 LP 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 LP antibody diluent of 2mg/ml by mouse-anti people LP 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 LP 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 LP 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 0.023ng/ml, accuracy (recovery) 99.24%, precision (CV) 3.06%, 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 LP 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 LP antibody diluent of 2mg/ml by mouse-anti people LP 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 LP 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 LP 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 0.8ng/ml, accuracy (recovery) 98.24%, precision (CV) 4.56%, stability 12 months.
According to its test result, detection kit provided by the present invention, has good sensitivity for analysis, accuracy, accuracy and stability.
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 (11)
1. the latex enhancing immune detecting LP, than a turbid kit, comprises 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 LP 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 LP antibody; damping fluid 1 is comprise 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 1 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; Damping fluid 2 in described reagent R2 is selected from one or more in PBS damping fluid, borate buffer solution, GOODS damping fluid, glycine buffer, and its pH is 7.0 ~ 9.0, and concentration is 25 ~ 500mmol/L.
2. detection kit as claimed in claim 1, it is characterized in that, described GOODS damping fluid is selected from one or more in Hepes damping fluid, MOPS damping fluid.
3. detection kit as claimed in claim 1, it is characterized in that, the surface functional group of the polystyrene latex microspheres in described reagent R2 is amino, carboxyl, hydrazides, aldehyde radical or epoxy radicals, and the particle diameter of polystyrene latex microspheres is at 100 ~ 600nm.
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%; Stabilizing agent 2 in described reagent R2 adopt ion stabilizer and suspension stabilizer with the use of; Wherein ion stabilizer is NaCl, KCl, Na
2cO
3, Na
2sO
4or K
2sO
4, suspension stabilizer is PEG8000, sucrose, glycerine or glucose.
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, is characterized in that, the polystyrene latex microspheres of described polystyrene latex microspheres to be surface functional group be carboxyl.
8. detection kit as claimed in claim 1, it is characterized in that, the LP antibody in described reagent R2 is one or more combination of mouse-anti people LP antibody, Goat anti human LPIgG antibody or the anti-human LPIgG antibody of rabbit.
9. 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).
10. detection kit as claimed in claim 1, it is characterized in that, described EDTA concentration is 10 ~ 100mmol/L.
The latex enhancing immune of the detection LP as described in 11. claims as arbitrary in claim 1-10, than the preparation method of turbid 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: LP antibody is carried out 4 DEG C of dialysis, then with damping fluid by LP antibody dilution to 2mg/ml, obtain LP 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 LP 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410736366.5A CN104459105B (en) | 2014-12-05 | 2014-12-05 | A kind of latex enhancing immune detecting LP is than turbid kit and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410736366.5A CN104459105B (en) | 2014-12-05 | 2014-12-05 | A kind of latex enhancing immune detecting LP is than turbid kit and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104459105A CN104459105A (en) | 2015-03-25 |
| CN104459105B true CN104459105B (en) | 2016-03-09 |
Family
ID=52905493
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410736366.5A Active CN104459105B (en) | 2014-12-05 | 2014-12-05 | A kind of latex enhancing immune detecting LP is than turbid kit and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104459105B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107478854B (en) * | 2017-08-10 | 2018-10-12 | 迈克生物股份有限公司 | A kind of lipoprotein a detection kit and detection method |
| CN109342724A (en) * | 2018-10-31 | 2019-02-15 | 廖朝晖 | A kind of kit detecting mycoplasma pneumoniae |
| CN111122892B (en) * | 2020-02-12 | 2023-12-26 | 上海泰辉生物科技有限公司 | Full-automatic immunoassay device and detection method based on turbidimetry detection |
| CN113447661A (en) * | 2021-07-01 | 2021-09-28 | 安徽大千生物工程有限公司 | Leptin latex enhanced turbidimetry detection kit and preparation and use method thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IL132313A0 (en) * | 1999-10-11 | 2001-03-19 | Yeda Res & Dev | Leptin assay |
| DE10353593A1 (en) * | 2003-11-17 | 2005-06-23 | Klinikum der Universität München Großhadern-Innenstadt | Leptin antagonist and method for the quantitative measurement of leptin |
| CN100362110C (en) * | 2005-11-22 | 2008-01-16 | 浙江大学 | Leptin and leptin receptor gene polymorphism detecting chip, and its preparing method and use |
| CN102901823A (en) * | 2012-10-26 | 2013-01-30 | 安徽安科生物工程（集团）股份有限公司 | Leptin detection kit |
-
2014
- 2014-12-05 CN CN201410736366.5A patent/CN104459105B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN104459105A (en) | 2015-03-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104360081B (en) | Cystatin C detection kit of a kind of improvement and preparation method thereof | |
| CN104407157B (en) | A kind of ion stabilizer and suspension stabilizer with the use of the latex enhancing immune of detection leptin than turbid kit | |
| CN104459105B (en) | A kind of latex enhancing immune detecting LP is than turbid kit and preparation method thereof | |
| CN102759631B (en) | The latex enhancing immune of a kind of quantitative detection Procalcitonin PCT is than turbid kit | |
| CN102955033B (en) | Kit for determining glycocholic acid in human blood | |
| CN104459139B (en) | A kind of D dimer latex of surface functional group that utilizes strengthens immunoturbidimetry detection kit | |
| CN104407156B (en) | Detect the latex enhancing immune of polysaccharide associated proteins (LBP) than turbid kit and preparation method thereof | |
| CN106932594A (en) | A kind of preferred cystatin C detection kit and preparation method thereof | |
| CN105067615B (en) | A kind of detection kit and detection method based on amide group nano rubber latex enhancing turbidimetry animal C reactive proteins | |
| CN104237522A (en) | Adiponectin content detection kit and preparation method thereof | |
| CN101903776B (en) | Method for measuring endotoxin and reagent kit for measuring endotoxin | |
| CN104360072A (en) | D-dimmer latex-reinforced immunonephelometry detection kit cooperatively adopting ion stabilizer and suspension stabilizer | |
| CN106198415A (en) | A kind of test kit measuring glycolated hemoglobin and preparation method thereof | |
| CN101479604A (en) | Kit for detection/quantification of analyte, and method for detection/quantification of analyte | |
| CN104391120B (en) | The latex enhancing immune of a kind of detection leptin utilizing surface functional group is than turbid test kit | |
| CN102636653A (en) | Compounded latex particle-enveloped cystatin C detection kit | |
| CN103308698A (en) | Method for covalently coupling amino-containing molecules to microspheres | |
| CN106018299A (en) | Kit for measuring serum amyloid A and preparation method of kit | |
| CN108931648A (en) | A kind of cystatin C detection kit based on latex immunoturbidimetry | |
| CN104360086B (en) | The latex enhancing immune of a kind of soluble leptin receptor is than turbid detection kit | |
| CN104535761B (en) | Hyperbranched polyglycidyl ether is modified latex microsphere and is strengthened immunoturbidimetry and application thereof | |
| CN104459153B (en) | A kind of sOB-R latex of surface functional group that utilizes strengthens immunoturbidimetry detection kit | |
| CN104535758A (en) | Method for coupling polypeptide or protein to microsphere in covalent mode | |
| CN106970226A (en) | The kit of the ECD concentration levels of HER 2 in a kind of measure human serum | |
| CN104360084A (en) | Ion stabilizing agent and suspension stabilizing agent cooperatively used kit for detecting sOB-R (soluble leptin receptor) by latex enhanced turbidimetric immunoassay |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20180105 Address after: 400000 Taikang Road Dadukou District of Chongqing Bridge Industrial Park C District No. 6 Building 30 layer 1-4 Patentee after: Chongqing Zhongyuan Ji Ji Biological Technology Co., Ltd. Address before: Jiulongpo Branch Park four street 400039 Chongqing city 70-1, 70-2, J block three layer Patentee before: CHONGQING ZHONGYUAN BIOTECHNOLOGY CO., LTD. |
|
| TR01 | Transfer of patent right | ||
| CP01 | Change in the name or title of a patent holder |
Address after: Floor 1-4, building 30, no.6, Taikang Road, Zone C, Jianqiao Industrial Park, Dadukou District, Chongqing 400000 Patentee after: Zhongyuan Huiji Biotechnology Co.,Ltd. Address before: Floor 1-4, building 30, no.6, Taikang Road, Zone C, Jianqiao Industrial Park, Dadukou District, Chongqing 400000 Patentee before: CHONGQING ZHONGYUAN HUIJI BIOTECHNOLOGY Co.,Ltd. |
|
| CP01 | Change in the name or title of a patent holder |