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 LP IgG antibody or the anti-human LP IgG 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 MOLECULAR CLONING:A LABORATORY MANUAL such as Sambrook, Second edition, Cold Spring HarborLaboratory Press, 1989 and Third edition, 2001; Ausubel etc., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; The seriesMETHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATINSTRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODSIN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), AcademicPress, San Diego, 1999; With METHODS IN MOLECULAR BIOLOGY, Vol.119, ChromatinProtocols (P.B.Becker, ed.) Humana Press, 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, 20g NaCl, 50g PEG8000,0.5g Sodium azide, 25g bovine serum albumin(BSA) and 14.2g EDTA 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, 40g NaCl 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, 9g NaCl, 15g PEG8000,0.5g Sodium azide, 5g bovine serum albumin(BSA) and 14.5g EDTA 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, 9g NaCl 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, 9g NaCl, 15g PEG8000,0.5g Sodium azide, 5g bovine serum albumin(BSA) and 14.5g EDTA 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, 9g NaCl 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.