CN104360056A - Method for improving sensitivity of latex enhanced turbidimetric immunoassay - Google Patents
Method for improving sensitivity of latex enhanced turbidimetric immunoassay Download PDFInfo
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Abstract
The invention relates to the field of in-vitro diagnosing reagents, and particularly relates to a method for improving the sensitivity of latex enhanced turbidimetric immunoassay. The method comprises the following steps: (1) selecting latex particles with large diameters to react with an antibody, and carrying out antibody coating; (2) washing and sealing the coated latex, dispersing the latex in a buffer solution to prepare a reagent 2 of a latex enhanced turbidimetric immunoassay kit; (3) preparing a reagent 1 of the latex enhanced turbidimetric immunoassay kit; (4) setting parameters on a full-automatic biochemical analyzer; (5) operating the biochemical analyzer to calibrate a sample with known concentration and detect an absorbance change value of the sample to be detected, and calculating the content of to-be-detected object in the sample according to the calibration curve. The method for improving the sensitivity of latex enhanced turbidimetric immunoassay is capable of greatly improving the analyzing sensitivity and precision of a reagent without increasing the detection cost, and has an excellent application prospect.
Description
Technical field
The present invention relates to external diagnosis reagent field, particularly relate to a kind of method promoting the sensitivity of latex enhancing immune turbidimetry.
Background technology
When light by a turbid media solution time, owing to there is muddy particle in solution, light is absorbed a part, absorption number be directly proportional to the amount of muddy particle, the method for this mensuration absorbing amount is called turbidimetry.This method is applied to after plasma proteins is combined with its antibody early than reports such as nineteen fifty-nine Schultre and Schuick and forms compound, cause the change of turbidity, carry out transmission turbidimetric assay again, the general antibody that adopts, to the turbidimetry of antigen quantify, is called Immunity transmission turbidity.Its principle is, utilize antigen and antibody specific binding formed compound, by measure complex formation number quantitative method is carried out to antigen or antibody.
But the immunoturbidimetry of classics, a small amount of little antigen antibody complex is extremely difficult forms turbidity, unless placed the long period; As formed larger compound, then antigen and antibody consumption are also comparatively large, obviously do not meet the requirement of milligram ammonia.So developed the latex enhancing immune turbidimetry (PETIA) being widely used in now Biochemical Analyzer: the improvement immunoturbidimetry analytic approach based on polyclonal antibody, gene engineering method is utilized to be combined with latex particle by antibody, just Ag-Ab-latex particle compound is defined when antigen-antibody combines, enhance reaction absorbance, reactant liquor at a given wavelength than turbid, compare with the same titer processed, calculate the content of antigen in sample.Utilize Biochemical Analyzer to carry out turbidimetric assay, whole analytic process only needs a few minutes, and the method compares its sensitivity with traditional immunization turbidimetry higher.
Along with the development of laboratory medicine, detection for low concentration detection material gets more and more, the concentration of detection material drops to ng/mL rank from μ g/mL rank, sensitivity requirement for diagnostic reagent is also more and more higher, need further to promote the sensitivity detected for traditional latex enhancing immune turbidimetry, to adapt to testing requirement, the method of traditional lifting detection sensitivity comprises the antibody adopting affinity higher, adopt larger particle, the better latex particle of stability, select the buffer solution system etc. better promoting antigen-antibody reaction, but these all can increase the cost of reagent, how to promote the sensitivity for analysis in the face of ng/mL rank analyte and precision, do not increase a major challenge that cost is reagent exploitation always simultaneously.
Summary of the invention
The shortcoming of prior art in view of the above, the object of the invention is to the selection by reacting wavelength, not only detecting the generation of newborn antigen-antibody complex, also detecting the minimizing in conjunction with latex particle, improve sensitivity for analysis and the precision of detection material, 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 method promoting the sensitivity of latex enhancing immune turbidimetry, comprises the steps:
1) select latex particle and the antibody response of Large stone, carry out antibody bag quilt, the particle diameter of the latex particle of Large stone is 0.2-1.0 μm;
2) by bag by after latex after cleaning and closed step, be scattered in damping fluid, obtained latex enhancing immune turbidimetry kit reagent 2;
3) damping fluid promoting antigen and antibody response effect is configured with, obtained latex enhancing immune turbidimetry kit reagent 1;
4) on automatic clinical chemistry analyzer, parameters, wherein, predominant wavelength is set to 300-500nm, and commplementary wave length is set to 500-800nm, and the Direction of Reaction is negative direction;
5) run biochemical instruments, after using the sample of concentration known to calibrate, detect sample to be tested and obtain absorbance changing value, according to calibration curve, calculate the content of determinand in sample.
Preferably, in described step 2, damping fluid is Tris-HCl damping fluid, and concentration is 50mM, and wherein the concentration of bovine serum albumin(BSA) BSA is 5g/L, and the concentration of Sodium azide is 1g/L.
Preferably, in described step 3, damping fluid is Tris-HCl damping fluid, and its formula is: the concentration of Tris 50mmol/L, bovine serum albumin(BSA) BSA is 3g/L, and the concentration of Sodium azide is the concentration of 1g/L, PEG6000 is 50g/L, stachyose 0.8-3g/L; Alum 0.1-1g/L; Fructose Diphosphate 0.8-3g/L; Sodium hexametaphosphate 0.05-0.5g/L, pH=7.0-7.4.
Preferably, the method for described lifting latex enhancing immune turbidimetry sensitivity is not for the purpose of the Diagnosis and Treat of disease.
Preferred, not obtain diagnostic result or health status to the direct object of the measurement of determinand content, and just obtain the method as the information of intermediate result from human body or animal body, or be that the tissue departing from human body or animal body, body fluid or excreta are processed or detect the method for the information obtained as intermediate result.
Second aspect present invention provides the purposes of method in field of biological detection of described lifting latex enhancing immune turbidimetry sensitivity.
Third aspect present invention provides a kind of high sensitivity latex enhancing immune turbidimetry kit, comprise reagent 1 and reagent 2, described reagent 2 is for being coated with the latex particle dispersion liquid of antibody, the particle diameter of described latex particle is 0.2-1.0 μm, and described reagent 1 is the damping fluid promoting antigen and antibody response.
Preferably, the formula of described reagent 1 is: Tris 50mmol/L, and the concentration of bovine serum albumin(BSA) BSA is 3g/L, and the concentration of Sodium azide is the concentration of 1g/L, PEG6000 is 50g/L, stachyose 0.8-3g/L; Alum 0.1-1g/L; Fructose Diphosphate 0.8-3g/L; Sodium hexametaphosphate 0.05-0.5g/L.
Preferably, in described reagent 1, pH value is neutral.
Preferably, in described reagent 2, the antibody of bag quilt is corresponding with detection thing.
Be more preferably DDi antibody.
Preferably, in described reagent 2, in dispersion liquid, the concentration of Tris-HCl damping fluid is 50mM, and the concentration of bovine serum albumin(BSA) BSA is 5g/L, and 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, comprises the steps:
1. select latex particle and the antibody response of Large stone, carry out antibody bag quilt, the particle diameter of described latex particle is 0.2-1.0 μm;
2. by bag by after latex after cleaning and closed step, be scattered in specific damping fluid, obtained latex enhancing immune turbidimetry kit reagent 2;
3. be configured with the damping fluid promoting antigen and antibody response effect, obtained latex enhancing immune turbidimetry kit reagent 1.Fifth aspect present invention provides described high sensitivity latex enhancing immune turbidimetry kit in the purposes of field of biological detection.
The present invention is according to the ultimate principle of latex enhancing immune turbidimetry: the latex and the antigen-reactive that are namely coated with antibody, form larger latex antigen antibody complex, cause the increase of turbidity, enhance reaction absorbance, reactant liquor is tested at a given wavelength, compare with the same titer processed, calculate the content of antigen in sample.The present inventor thinks when coated antibody latex particle particle diameter is larger, latex particle itself has certain turbidity, at specific wavelength, there is larger absorption, and after coated antibody latex and antigen form larger latex antigen antibody complex, for the coated antibody latex particle of reaction also reduces according to Mie theory thereupon, the microballoon of different-diameter is different for the luminous reflectanc of fixed wave length, so whole reactant liquor absorbance under some wavelength increases, but absorbance can decline under some wavelength, by measuring the absorbance changing value of rising wavelength and decline wavelength, and sum up, more than 1 times of sensitivity for analysis can be promoted.
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, by the damping fluid of special promotion antigen-antibody reaction, and utilize further Large stone bag reduced by latex microsphere caused by absorbance change and latex antigen antibody complex increase caused by absorbance change (Large stone bag reduced by latex microsphere caused by absorbance change be often greater than latex antigen antibody complex increase caused by absorbance change), the change of both absorbances add and after, the sensitivity for analysis of reagent 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 to be realized by the mode revising parameter on full automatic biochemical apparatus, so can not cost be increased, secondly due to large grain size latex microballoon bag by required antibody often fewer than the small grain size latex microballoon of homogenous quantities (surface area is less), the present invention use latex concentration required for the present invention less, so often can reduce the cost of reagent simultaneously.
Accompanying drawing explanation
Fig. 1 is the reaction full wavelength scanner dynamic experiment result of detection kit prepared by embodiment 1.
Fig. 2 is the calibration graph of detection kit prepared by embodiment 1.
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, 1987 and 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.
Need to illustrate, the present invention's raw material used can be substantially all commercially available prod, wherein trishydroxymethylaminomethane Tris available from Sigma; Wherein carboxylic polystyrene latex particle adds with the form of carboxylic polystyrene latex particle solution, purchased from Japanese JSR company; DDi antibody and DDi are purchased from hytest company of Finland.
Embodiment 1
One, the preparation of latex enhancing immune than turbid kit is quantitatively detected
The preparation of reagent 1:
By concentration be the Tris-HCl damping fluid of 50mM, 3g bovine serum albumin(BSA) BSA, 1g Sodium azide, 2.2g stachyose, 0.6g alum, 1.9g Fructose Diphosphate, 0.3g sodium hexametaphosphate and 50g PEG6000 prepare, in final reagent 1, the concentration of Tris-HCl damping fluid is 50mM, the concentration of bovine serum albumin(BSA) BSA is 3g/L, 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, sodium hexametaphosphate 0.3g/L;
Take 6.06g trishydroxymethylaminomethane (Tris), 3g BSA, 50g PEG6000, stachyose 2.2g, alum 0.6g, Fructose Diphosphate 1.9g, sodium hexametaphosphate 0.3g, 1g Sodium azide be dissolved in 0.8L deionized water, regulate pH to 7.0 with HCl, be settled to 1L and namely obtain reagent 1.
The preparation of reagent 2:
By concentration be the Tris-HCl damping fluid of 50mM, the Sodium azide of bovine serum albumin(BSA) BSA, 1g of 5g and bag prepared by the sensitization polystyrene latex particles of mouse-anti people DDi antibody, in final reagent 2, the concentration of Tris-HCl damping fluid is 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 the carboxylic polystyrene latex particle solution 100 μ L that mass volume ratio is 10%, adding mass concentration is wherein 1%EDAC solution 10 μ L, after being placed in 37 DEG C of shaking table reaction 0.5 ~ 1h, centrifuging 30min under the rotating speed of 12000rmp again, then supernatant liquor is outwelled, again with concentration to be 50mM, pH be 7.2 glycine buffer wash three times, finally by precipitation, to be scattered in 1L concentration be 50mM, pH is in the glycine buffer of 7.2, and the concentration (in mass volume ratio) making polystyrene latex particles in final solution is 0.5 ~ 1%.
2. the purifying of antibody:
Mouse-anti people DDi antibody is joined in bag filter, is dialyse 48 hours in the PBS damping fluid of 7.2 at 100mM, pH, in the process of dialysis, changes damping fluid 3 times, obtain the mouse-anti people DDi antibody of purifying.
3. the coupling of antibody latex particle:
Carboxylic polystyrene latex particle solution after 1. step activates mixed with the antibody after step 2. purifying, mixing is placed on 37 DEG C of shaking tables and hatches 2h, obtains antibody-latex particle compound; Then by antibody-latex particle compound at the centrifugal 30min of 12000rpm, outwell supernatant, again with concentration to be 100mM, pH be 7.2 PBS buffer solution 2 times, finally to add concentration be again 50mM, pH be 7.0 Tris-HCl damping fluid 10ml, be uniformly mixed.The Sodium azide of bovine serum albumin(BSA) BSA and 1mg containing 0.5mg in the Tris-HCl damping fluid of wherein 10ml; Described bag is 0.05 ~ 0.20% by the mass volume ratio final concentration of the sensitization polystyrene latex particles of mouse-anti people DDi antibody.
The preparation of calibration object:
By concentration be the PBS damping fluid of 100mM, 3g bovine serum albumin(BSA) BSA, 1g Sodium azide and DDi to prepare, in final calibration object, the concentration of PBS damping fluid is 100mM, and the concentration of bovine serum albumin(BSA) BSA is 3g/L, and the concentration of Sodium azide is 1g/L;
Take 35.61Na
2hPO
42H
2o, 3g bovine serum albumin(BSA) BSA, 1g Sodium azide is dissolved in 0.8L deionized water, regulate pH to 7.2,1L is settled to distilled water, DDi is dissolved in the above-mentioned solution prepared, the concentration being mixed with DDi is respectively 0,0.5,1,2,4, the solution of 8ng/mL, namely obtain calibration object.
Two, the reaction full wavelength scanner dynamic experiment of detection kit
Calibration object is mixed with reagent 1,37 DEG C hatch 5min after, add reagent 2,37 DEG C hatch 10S after use ultraviolet spectrophotometer full wavelength scanner reactant liquor (for the first time), use ultraviolet spectrophotometer full wavelength scanner reactant liquor (for the second time) after 37 DEG C of reaction 4min50S, calculate absorbance changing value △ A=A2-A1; The wavelength 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 reduced value at 340nm place, is greater than its absorbance increase at 600nm place, if the change of the absorbance at two wavelength places is added and, greatly can promote the sensitivity for analysis of reagent.
Three, the using method of latex enhancing immune than turbid kit is quantitatively detected
Detecting instrument: OLYMPUS AU640 automatic clinical chemistry analyzer
Parameter arranged according to the present invention is: detection method: END. measures wavelength: predominant 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 is calibrated; The Direction of Reaction: negative.
The preparation of reagent 1:
By concentration be the Tris-HCl damping fluid of 50mM, 3g bovine serum albumin(BSA) BSA, 1g Sodium azide, 2.2g stachyose, 0.6g alum, 1.9g Fructose Diphosphate, 0.3g sodium hexametaphosphate and 50g PEG6000 prepare, in final reagent 1, the concentration of Tris-HCl damping fluid is 50mM, the concentration of bovine serum albumin(BSA) BSA is 3g/L, 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, sodium hexametaphosphate 0.3g/L;
Take 6.06g trishydroxymethylaminomethane (Tris), 3g BSA, 50g PEG6000, stachyose 2.2g, alum 0.6g, Fructose Diphosphate 1.9g, sodium hexametaphosphate 0.3g, 1g Sodium azide be dissolved in 0.8L deionized water, regulate pH to 7.0 with HCl, be settled to 1L and namely obtain reagent 1.
The preparation of reagent 2:
By concentration be the Tris-HCl damping fluid of 50mM, the Sodium azide of bovine serum albumin(BSA) BSA, 1g of 5g and bag prepared by the sensitization polystyrene latex particles of mouse-anti people DDi antibody, in final reagent 2, the concentration of Tris-HCl damping fluid is 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: predominant wavelength 600nm, the long nothing of negative wave; Sample size: 25 μ L; Reagent 1:240 μ L; Reagent 2:80 μ L; Calibrating mode: multiple spot is calibrated; The Direction of Reaction: just.
The preparation of contrast agent 1:
By concentration be the Tris-HCl damping fluid of 50mM, 3g bovine serum albumin(BSA) BSA, 1g Sodium azide and 50g PEG6000 prepare, in final reagent 1, the concentration of Tris-HCl damping fluid is 50mM, the concentration of bovine serum albumin(BSA) BSA is 3g/L, the concentration of Sodium azide is the concentration of 1g/L, PEG6000 is 50g/L;
Take 6.06g trishydroxymethylaminomethane (Tris), 3g BSA, 50g PEG6000,1g Sodium azide be dissolved in 0.8L deionized water, regulate pH to 7.0 with HCl, be settled to 1L and namely obtain reagent 1.
Reagent 2 is same as described above.
Calibration object is mixed with reagent 1,37 DEG C hatch 5min after, add reagent 2,37 DEG C hatch 10S after read absorbance A1, read absorbance A2 after reaction 4min50S, calculate absorbance changing value △ A=A2-A1; Then with △ A value for ordinate, corresponding calibration object concentration is horizontal ordinate, and draw 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 calculating reagent the results are shown in Table 2.Table is depicted as the sensitivity for analysis of parameters of the present invention and reduced parameter.
Table 2
Precision Experiment, adopt senior middle school's low value sample repeated test 10 times, characterize the precision of reagent with the imprecision of test result, result sees table 3.
Table 3
Sensitivity for analysis and the precision of the reagent of parameter of the present invention are all obviously better than reduced parameter, but cost does not increase.
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 (6)
1. promote a method for latex enhancing immune turbidimetry sensitivity, comprise the steps:
1) select latex particle and the antibody response of Large stone, carry out antibody bag quilt, the particle diameter of the latex particle of Large stone is 0.2-1.0 μm;
2) by bag by after latex after cleaning and closed step, be scattered in damping fluid, obtained latex enhancing immune turbidimetry kit reagent 2;
3) damping fluid promoting antigen and antibody response effect is configured with, obtained latex enhancing immune turbidimetry kit reagent 1;
4) on automatic clinical chemistry analyzer, parameters, wherein, predominant wavelength is set to 300-500nm, and commplementary wave length is set to 500-800nm, and the Direction of Reaction is negative direction;
5) run biochemical instruments, after using the sample of concentration known to calibrate, detect sample to be tested and obtain absorbance changing value, according to calibration curve, calculate the content of determinand in sample.
2. the method promoting the sensitivity of latex enhancing immune turbidimetry as claimed in claim 1, is characterized in that, in described step 2, damping fluid is Tris-HCl damping fluid, concentration is 50mM, and wherein the concentration of bovine serum albumin(BSA) BSA is 5g/L, and the concentration of Sodium azide is 1g/L.
3. the method promoting the sensitivity of latex enhancing immune turbidimetry as claimed in claim 1, it is characterized in that, in described step 3, damping fluid is Tris-HCl damping fluid, concentration is 50mM, and wherein 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 0.8-3g/L; Alum 0.1-1g/L; Fructose Diphosphate 0.8-3g/L; Sodium hexametaphosphate 0.05-0.5g/L.
4. the method promoting the sensitivity of latex enhancing immune turbidimetry as claimed in claim 1, it is characterized in that, the method for described lifting latex enhancing immune turbidimetry sensitivity is not for the purpose of the Diagnosis and Treat of disease.
5. the method promoting the sensitivity of latex enhancing immune turbidimetry as claimed in claim 4, it is characterized in that, not obtain diagnostic result or health status to the direct object of the measurement of determinand content, and just obtain the method as the information of intermediate result from human body or animal body, or be that the tissue departing from human body or animal body, body fluid or excreta are processed or detect the method for the information obtained as intermediate result.
6. the method for the lifting latex enhancing immune turbidimetry sensitivity as described in claim as arbitrary in claim 1-5 is in the purposes of field of biological detection.
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| CN110296944A (en) * | 2019-04-30 | 2019-10-01 | 宁波普瑞柏生物技术股份有限公司 | A method of improving immunoturbidimetry detection sensitivity and linear |
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| CN111830260A (en) * | 2020-07-27 | 2020-10-27 | 北京安图生物工程有限公司 | Process for reducing batch-to-batch difference of latex enhanced immunoturbidimetric reagent |
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