A kind of method that promotes the sensitivity of latex enhancing immune turbidimetry
Technical field
The present invention relates to external diagnosis reagent field, particularly relate to a kind of latex enhancing immune turbidimetry sensitivity that promotesMethod.
Background technology
In the time that light passes through a turbid media solution, owing to there is muddy particle in solution, light is absorbed a part,Absorb number be directly proportional to the amount of muddy particle, the method for this mensuration absorbing amount is called turbidimetry. This methodBe applied to plasma protein early than reports such as nineteen fifty-nine Schultre and Schuick and be combined rear formation compound with its antibody, causeThe change of turbidity, then carry out transmission turbidimetric assay, generally adopt the turbidimetry of antibody to antigen quantify, be called immune transmissionTurbidimetry. Its principle is to utilize the specific binding of antigen and antibody to form compound, by measuring the many of compound formation volumeLess antigen or antibody are carried out to quantitative method.
But classical immunoturbidimetry, the extremely difficult turbidity that forms of a small amount of little antigen antibody complex, unless placedFor a long time; As formed larger compound, antigen and antibody consumption are also larger, obviously do not meet the requirement of traceization. SoDevelop the latex enhancing immune turbidimetry (PETIA) that is widely used in now Biochemical Analyzer: taking polyclonal antibody as basisImprovement immunoturbidimetry analytic approach, utilize gene engineering method that antibody is combined with latex particle, in the time that antigen-antibody combinesJust formed Ag-Ab-latex particle compound, strengthened reaction absorbance, reactant liquor at certain wavelength place than turbid, withThe titer comparison of sample processing, the content of antigen in calculating sample. Utilize Biochemical Analyzer to carry out turbidimetric assay, whole analysisJourney only needs a few minutes, and relatively its sensitivity is higher for the method and traditional immunoturbidimetry.
Along with the development of laboratory medicine, more and more for the detection of low concentration detection material, the concentration of detection material justDropping to ng/mL rank from μ g/mL rank, also more and more higher for the sensitivity requirement of diagnostic reagent, for traditional glueBreast strengthens immunoturbidimetry needs further to promote the sensitivity detecting, to adapt to testing requirement, and traditional lifting inspectionThe method of surveying sensitivity comprises the higher antibody of employing affinity, adopts larger particle, and the better latex particle of stability, selectsBetter promote the buffer solution system of antigen-antibody reaction etc., but these all can increase the cost of reagent, how to promote and faceSensitivity for analysis and the precision of ng/mL rank analyte, do not increase cost is simultaneously a major challenge of reagent exploitation always.
Summary of the invention
The shortcoming of prior art in view of the above, the object of the invention is to by the selection of reaction wavelength, not only inspectionSurvey the generation of newborn antigen-antibody complex, also detect the not minimizing in conjunction with latex particle, improve the sensitivity for analysis of detection materialAnd precision, for solving the problems of the prior art.
For achieving the above object and other relevant objects, first aspect present invention provides a kind of latex enhancing immune ratio that promotesThe method of turbid method sensitivity, comprises the steps:
1) select latex particle and the antibody response of large particle diameter, carry out antibody coated, the particle diameter of the latex particle of large particle diameterFor 0.2-1.0 μ m;
2) by the latex after coated through cleaning and sealing step after, be scattered in buffer solution, make latex enhancing immuneTurbidimetry kit reagent 2;
3) dispose the buffer solution that promotes antigen and antibody response effect, make the examination of latex enhancing immune turbidimetry kitAgent 1;
4) on automatic clinical chemistry analyzer, parameters, wherein, dominant wavelength is set to 300-500nm, commplementary wave length settingFor 500-800nm, the Direction of Reaction is negative direction;
5) operation biochemical instruments, after the sample that uses concentration known is calibrated, detects sample to be tested and obtains absorbance changeChange value, according to calibration curve, calculates the content of determinand in sample.
Preferably, in described step 2, buffer solution is Tris-HCl buffer solution, and concentration is 50mM, wherein bovine serum albumin(BSA)The concentration of BSA is 5g/L, and the concentration of Sodium azide is 1g/L.
Preferably, in described step 3, buffer solution is Tris-HCl buffer solution, and its formula is: Tris50mmol/L, ox bloodThe concentration of pure protein B SA is 3g/L, and the concentration of Sodium azide is 1g/L, and the concentration of PEG6000 is 50g/L, stachyose 0.8-3g/L; Alum 0.1-1g/L; Fructose Diphosphate 0.8-3g/L; Calgon 0.05-0.5g/L, pH=7.0-7.4.
Preferably, the method for described lifting latex enhancing immune turbidimetry sensitivity be not taking the diagnosis of disease and treatment asObject.
Preferred, the direct object of the measurement to determinand content is not to obtain diagnostic result or health status, and onlyTo obtain the method as the information of intermediate object program from human body or animal body, or just to departing from the group of human body or animal bodyKnit, body fluid or excreta process or detect to obtain the method as the information of intermediate object program.
Second aspect present invention provides the method for described lifting latex enhancing immune turbidimetry sensitivity to lead in biological detectionThe purposes in territory.
Third aspect present invention provides a kind of high sensitivity latex enhancing immune turbidimetry kit, comprises reagent 1 and examinationAgent 2, described reagent 2 is for being coated with the latex particle dispersion liquid of antibody, and the particle diameter of described latex particle is 0.2-1.0 μ m, described inReagent 1 is for promoting the buffer solution of antigen and antibody response.
Preferably, the formula of described reagent 1 is: Tris50mmol/L, the concentration of bovine serum albumin(BSA) BSA is 3g/L, foldedThe concentration of nitrogen sodium is 1g/L, and the concentration of PEG6000 is 50g/L, stachyose 0.8-3g/L; Alum 0.1-1g/L; Fructose diphosphateSodium 0.8-3g/L; Calgon 0.05-0.5g/L.
Preferably, in described reagent 1, pH value is neutral.
Preferably, in described reagent 2, coated antibody is corresponding with detection thing.
More preferably DDi antibody.
Preferably, in described reagent 2, in dispersion liquid, the concentration of Tris-HCl buffer solution is 50mM, bovine serum albumin(BSA) BSAConcentration be 5g/L, the concentration of Sodium azide is 1g/L.
Fourth aspect present invention provides the preparation method of described high sensitivity latex enhancing immune turbidimetry kit, comprisesFollowing steps:
1. select latex particle and the antibody response of large particle diameter, carry out antibody and be coated with, the particle diameter of described latex particle is0.2-1.0μm;
By the latex after coated through cleaning and sealing step after, be scattered in specific buffer solution, make latex intensifiedImmunoturbidimetry kit reagent 2;
3. dispose the buffer solution that promotes antigen and antibody response effect, make the examination of latex enhancing immune turbidimetry kitAgent 1. Fifth aspect present invention provides the use of described high sensitivity latex enhancing immune turbidimetry kit in field of biological detectionOn the way.
The present invention is according to the general principle of latex enhancing immune turbidimetry: be coated with latex and the antigen-reactive of antibody,Form larger latex antigen antibody complex, cause the increase of turbidity, strengthened reaction absorbance, reactant liquor is at certain wavelengthPlace's test, with the titer comparison of same processing, calculates the content of antigen in sample. The inventor thinks at coated antibody latexIn the larger situation of grain diameter, latex particle itself has certain turbidity, has larger absorption, and work as at specific wavelengthCoated antibody latex and antigen form after larger latex antigen antibody complex, for the coated antibody latex particle of reaction also withMinimizing according to Mie theory, the microballoon of different-diameter is for the luminous reflectanc difference of fixed wave length, so whole anti-Answer liquid absorbance under some wavelength to increase, but absorbance can decline under some wavelength, by measure rising wavelength and underFall the absorbance changing value of wavelength, and sum up, can promote the more than 1 times of sensitivity for analysis.
Compared with prior art, the following advantage of tool of the present invention:
1. greatly promote sensitivity for analysis and the precision of reagent: inventor finds, anti-by special promotionThe buffer solution of antigen-antibody reaction, and further utilize the coated latex microsphere of large particle diameter to reduce caused absorbance variation and latexAntigen antibody complex increases caused absorbance variation, and (the coated latex microsphere of particle diameter reduces caused absorbance variation greatlyOften being greater than latex antigen antibody complex increases caused absorbance and changes), both absorbances change add and after, reagentSensitivity for analysis promote one times or more than, the meanwhile lifting of sensitivity for analysis also brings the lifting of precision.
2. can not increase or reduce cost: first the present invention is only the change of metering system, can be easy toOn full automatic biochemical apparatus, realize by the mode of amendment parameter, thus cost can not increased, secondly because large grain size latex is micro-Bag often than the small grain size latex microballoon of homogenous quantities few (surface area is less), is used required for the present invention by required antibody simultaneouslyLatex concentration is less, so the present invention often can reduce the cost of reagent.
Brief description of the drawings
Fig. 1 is the reaction full wavelength scanner dynamic experiment result of the detection kit prepared of embodiment 1.
Fig. 2 is the calibration graph of the detection kit prepared of embodiment 1.
Detailed description of the invention
Below, by specific instantiation explanation embodiments of the present invention, those skilled in the art can be by this descriptionDisclosed content is understood other advantages of the present invention and effect easily. The present invention can also be by other different concrete realityThe mode of executing is implemented or is applied, and the every details in this description also can, based on different viewpoints and application, not deviate fromUnder spirit of the present invention, carry out various modifications or change.
Before further describing the specific embodiment of the invention, should be understood that protection scope of the present invention is not limited to downState specific specific embodiments; It is also understood that the term using in the embodiment of the present invention is specific concrete in order to describeEmbodiment, instead of in order to limit the scope of the invention; In description of the present invention and claims, unless in literary compositionExplicitly point out in addition, singulative " ", " one " and " this " comprise plural form.
In the time that embodiment provides number range, unless should be understood that the present invention is otherwise noted, two ends of each number rangeBetween point and two end points, any one numerical value all can be selected. Unless otherwise defined, all technology that use in the present invention andScientific terminology is identical with the meaning that those skilled in the art of the present technique understand conventionally. Except the concrete grammar using in embodiment, equipment,Outside material, the grasp according to those skilled in the art to prior art and record of the present invention, can also be used and thisMethod described in inventive embodiments, equipment, material any method, equipment and the material similar or prior art that is equal to come realExisting the present invention.
Unless otherwise indicated, in the present invention, disclosed experimental technique, detection method, preparation method all adopt this technology neckMolecular biology, biochemistry, chromatin Structure and the analysis of territory routine, analytical chemistry, cell cultivation, recombinant DNA technology andThe routine techniques of association area. These technology are existing in existing document improves explanation, specifically can be referring to Sambrook etc.MOLECULARCLONING:ALABORATORYMANUAL,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.
Need to illustrate, the present invention's raw material used can be substantially all commercially available prod, wherein trihydroxy methyl ammoniaMethylmethane Tris is purchased from Sigma company; Wherein carboxylic polystyrene latex particle is molten with carboxylic polystyrene latex particleThe form of liquid adds, purchased from Japanese JSR company; DDi antibody and DDi are purchased from hytest company of Finland.
Embodiment 1
One, quantitatively detect latex enhancing immune than the preparation of turbid kit
The preparation of reagent 1:
The Tris-HCl buffer solution that is 50mM by concentration, 3g bovine serum albumin(BSA) BSA, 1g Sodium azide, 2.2g stachyose,0.6g alum, 1.9g Fructose Diphosphate, 0.3g calgon and 50gPEG6000 prepare, in final reagent 1The concentration of Tris-HCl buffer solution is 50mM, and the concentration of bovine serum albumin(BSA) BSA is 3g/L, and the concentration of Sodium azide is 1g/L,The concentration of PEG6000 is 50g/L, stachyose 2.2g/L, alum 0.6g/L, Fructose Diphosphate 1.9g/L, calgon0.3g/L;
Take 6.06g trishydroxymethylaminomethane (Tris), 3gBSA, 50gPEG6000, stachyose 2.2g, alum0.6g, Fructose Diphosphate 1.9g, calgon 0.3g, 1g Sodium azide are dissolved in 0.8L deionized water, with HCl regulate pH extremely7.0, be settled to 1L and obtain reagent 1.
The preparation of reagent 2:
The Sodium azide of bovine serum albumin(BSA) BSA, the 1g of the Tris-HCl buffer solution that is 50mM by concentration, 5g and coated mouse-antiThe sensitization polystyrene latex particle of people's DDi antibody prepares, the concentration of Tris-HCl buffer solution in final reagent 2For 50mM, the concentration of bovine serum albumin(BSA) BSA is 5g/L, and the concentration of Sodium azide is 1g/L;
1. the activation of newborn particle, washing:
Get mass volume ratio and be 10% carboxylic polystyrene latex particle solution 100 μ L, add wherein quality denseDegree is 1%EDAC solution 10 μ L, and being placed in 37 DEG C of shaking tables reacts after 0.5~1h, then centrifugation under the rotating speed of 12000rmp30min, then outwells supernatant liquor, then is that 50mM, pH are 7.2 glycine buffer washing three times by concentration, finally will sinkIt is that 50mM, pH are in 7.2 glycine buffer that shallow lake is scattered in 1L concentration, makes the dense of polystyrene latex particle in final solutionDegree (taking mass volume ratio) is 0.5~1%.
2. the purifying of antibody:
Mouse-anti people DDi antibody is joined in bag filter, in the PBS buffer solution that is 7.2 at 100mM, pH, dialyse 48Hour, in the process of dialysis, change buffer solution 3 times, obtain the mouse-anti people DDi antibody of purifying.
3. the coupling of antibody latex particle:
By the carboxylic polystyrene latex particle solution after 1. step activates and step, 2. the antibody after purifying is mixedClose, mix and be placed on 37 DEG C of shaking tables and hatch 2h, obtain antibody-latex particle compound; Then by antibody-latex particle compoundAt the centrifugal 30min of 12000rpm, outwell supernatant, then be that 100mM, pH are 7.2 PBS buffer solution washing 2 times by concentration, lastAdding concentration is that 50mM, pH are 7.0 Tris-HCl buffer solution 10ml again, is uniformly mixed. The wherein Tris-of 10mlIn HCl buffer solution, contain the bovine serum albumin(BSA) BSA of 0.5mg and the Sodium azide of 1mg; Described coated mouse-anti people DDi antibodyThe mass volume ratio final concentration of sensitization polystyrene latex particle be 0.05~0.20%.
The preparation of calibration object:
The PBS buffer solution that is 100mM by concentration, 3g bovine serum albumin(BSA) BSA, 1g Sodium azide and DDi are to be prepared intoArrive, in final calibration object, the concentration of PBS buffer solution is 100mM, and the concentration of bovine serum albumin(BSA) BSA is 3g/L, Sodium azide denseDegree is 1g/L;
Take 35.61Na2HPO4·2H2O, 3g bovine serum albumin(BSA) BSA, 1g Sodium azide are dissolved in 0.8L deionized water, adjustJoint pH to 7.2, is settled to 1L with distilled water, DDi is dissolved in the above-mentioned solution preparing, and is mixed with DDiConcentration is respectively 0,0.5,1,2,4, the solution of 8ng/mL, obtains calibration object.
Two, the reaction full wavelength scanner dynamic experiment of detection kit
Calibration object is mixed with reagent 1, hatch after 5min for 37 DEG C, add 2,37 DEG C of reagent to hatch to use after 10S purpleOuter spectrophotometer full wavelength scanner reactant liquor (for the first time), uses ultraviolet specrophotometer all-wave after 37 DEG C of reaction 4min50SLong scan reactant liquor (for the second time), calculates absorbance changing value △ A=A2-A1; The wave-length coverage of full wavelength scanner is 200-800nm. Table 1 is depicted as full wavelength scanner dynamic experiment result.
Table 1
Can see, reactant liquor, in the absorbance minimizing value at 340nm place, is greater than its absorbance value added at 600nm place,If by the absorbance at two wavelength places change add and, can greatly promote the sensitivity for analysis of reagent.
Three, quantitatively detect latex enhancing immune than the using method of turbid kit
Detecting instrument: OLYMPUSAU640 automatic clinical chemistry analyzer
Parameter arranged according to the present invention is: detection method: END. measures wavelength: dominant wavelength 340nm, the long 600nm of negative wave;Sample size: 25 μ L; Reagent 1:240 μ L; Reagent 2:80 μ L; Calibrating mode: multiple spot calibration; The Direction of Reaction: negative.
The preparation of reagent 1:
The Tris-HCl buffer solution that is 50mM by concentration, 3g bovine serum albumin(BSA) BSA, 1g Sodium azide, 2.2g stachyose,0.6g alum, 1.9g Fructose Diphosphate, 0.3g calgon and 50gPEG6000 prepare, in final reagent 1The concentration of Tris-HCl buffer solution is 50mM, and the concentration of bovine serum albumin(BSA) BSA is 3g/L, and the concentration of Sodium azide is 1g/L,The concentration of PEG6000 is 50g/L, stachyose 2.2g/L, alum 0.6g/L, Fructose Diphosphate 1.9g/L, calgon0.3g/L;
Take 6.06g trishydroxymethylaminomethane (Tris), 3gBSA, 50gPEG6000, stachyose 2.2g, alum0.6g, Fructose Diphosphate 1.9g, calgon 0.3g, 1g Sodium azide are dissolved in 0.8L deionized water, with HCl regulate pH extremely7.0, be settled to 1L and obtain reagent 1.
The preparation of reagent 2:
The Sodium azide of bovine serum albumin(BSA) BSA, the 1g of the Tris-HCl buffer solution that is 50mM by concentration, 5g and coated mouse-antiThe sensitization polystyrene latex particle of people's DDi antibody prepares, the concentration of Tris-HCl buffer solution in final reagent 2For 50mM, the concentration of bovine serum albumin(BSA) BSA is 5g/L, and the concentration of Sodium azide is 1g/L;
Reduced parameter is: detection method: END. measures wavelength: dominant wavelength 600nm, the long nothing of negative wave; Sample size: 25 μ L; ExaminationAgent 1:240 μ L; Reagent 2:80 μ L; Calibrating mode: multiple spot calibration; The Direction of Reaction: just.
The preparation of contrast agent 1:
The Tris-HCl buffer solution that is 50mM by concentration, 3g bovine serum albumin(BSA) BSA, 1g Sodium azide and 50gPEG6000Prepare, in final reagent 1, the concentration of Tris-HCl buffer solution is 50mM, and the concentration of bovine serum albumin(BSA) BSA is 3g/L, foldedThe concentration of nitrogen sodium is 1g/L, and the concentration of PEG6000 is 50g/L;
Taking 6.06g trishydroxymethylaminomethane (Tris), 3gBSA, 50gPEG6000,1g Sodium azide is dissolved in 0.8L and goesIn ionized water, with HCl adjusting pH to 7.0, be settled to 1L and obtain reagent 1.
Reagent 2 is same as described above.
Calibration object is mixed with reagent 1, hatch after 5min for 37 DEG C, read suction after adding 2,37 DEG C of reagent to hatch 10SShading value A1, reads absorbance A2 after reaction 4min50S, calculates absorbance changing value △ A=A2-A1; Then taking △ A value asOrdinate, corresponding calibration object concentration is abscissa, draws calibration curve, calibration curve is as shown in Figure 2.
Four, latex enhancing immune is than the sensitivity for analysis of turbid kit and precision contrast experiment
According to calibration curve, the sensitivity for analysis that calculates reagent the results are shown in Table 2. Table is depicted as parameters of the present invention and rightThan the sensitivity for analysis of parameter.
Table 2
Precision Experiment, adopts senior middle school's low value sample retest 10 times, characterizes examination with the imprecision of test resultThe precision of agent, result is seen table 3.
Table 3
The sensitivity for analysis of the reagent of parameter of the present invention and precision are all obviously better than reduced parameter, but cost is notIncrease.
In sum, the present invention has effectively overcome 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 ripeThe personage who knows this technology all can, under spirit of the present invention and category, modify or change above-described embodiment. CauseThis, have in technical field under such as conventionally know the knowledgeable do not depart under disclosed spirit and technological thought completeAll equivalences that become are modified or change, and must be contained by claim of the present invention.