CN105510604A - Method for improving sensitivity and linearity of latex reagent - Google Patents
Method for improving sensitivity and linearity of latex reagent Download PDFInfo
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Abstract
The invention provides a method for improving the sensitivity and linearity of a latex reagent. An adopted detection kit comprises a reagent R1 and a reagent R2. The method is characterized in that the latex reagent is a latex reagent mixed with latexes with large and small particle sizes, and is processed to prepare a C-creative protein latex turbidimetric detection kit, and the kit comprises the reagent R1 and the reagent R2. The method comprises the following steps: (1) directionally coating latex microspheres with large particle sizes with mouse to-be-tested antigen monoclonal antibodies; (2) directionally coating latex microspheres with small particle sizes with rabbit or sheep to-be-tested antigen monoclonal antibodies; (3) mixing the coated latex microspheres with the large particle sizes and the coated latex microspheres with the small particle sizes.
Description
Technical field
The invention belongs to field of medical examination, be specifically related to a kind of mixed size grain size latex and improve emulsion reagent sensitivity and linear method simultaneously.
Background technology
C reactive protein (CRP) is a kind of Acute reaction protein, produces and is synthesized by liver cell, the serumβ globulin that molecular weight is 115-140KD, half life is 19 days after body is subject to the struvite stimulations such as microorganism invasion or tissue damage in several hours; All can measure in the multiple body fluid such as serum, cerebrospinal fluid, Pleural effusions of people.The detection of CRP is quite extensive in clinical practice, comprises the diagnosis and differential diagnosis of acute infectious diseases, the monitoring of post-operative infection; The observation of microbiotic curative effect; Course of disease detection and Index for diagnosis etc.The method of routine clinical mensuration CRP can detect the concentration of CRP>10mg/L when infection or tissue damage, but for want of higher sensitivity, well can not detect the CRP change of low concentration (0.1-10mg/L), and the CRP of this concentration and neonatal infection, angiocardiopathy have close relationship, namely usually said super quick CRP (hsCRP).In recent years, along with the development of inspection technology, start to have occurred a kind of full-range C RP, this CRP can meet higher sensitivity, can meet again higher linear, but be limited to the restriction of latex turbidimetric technic platform, these full-range Cs RP often or reducing susceptibility obtains High Linear, or reduction linearly obtains high sensitivity, therefore, existing latex is optimized than turbid platform technology and is improved, to meet sensitivity and High Linear just becomes a urgent problem simultaneously.
The method of mensuration CRP concentration conventional at present has fluorescence immune chromatography method, colloidal gold method, enzyme linked immunosorbent assay, latex enhancing immune turbidimetry etc.Although wherein fluorescence immune chromatography method, colloidal gold method, enzyme linked immunosorbent assay part can solve sensitivity and linear problem simultaneously, can only accomplish qualitative or sxemiquantitative, especially in super quick CRP concentration range, measured value accuracy is very low; Conventional latex enhancing immune turbidimetry measured value is accurate, but cannot take into account high sensitivity and the wide range of linearity.
Latex immunoturbidimetry is divided into turbidimetry and scattered light urbidmetry.The former is for Biochemical Analyzer, and the latter is used for specific protein analyser.Biochemical Analyzer feature is concentrated Sample, and with duration, but flux is high, and cost is low.Specific protein analyser, than Biochemical Analyzer compact, detects consuming time shorter, can go out result in several minutes, and be specially adapted to Outpatient and emergency and bedside monitoring use, but can not process sample in enormous quantities, cost is high.Execute in national and Xu Fei " c reactive protein rapid quantitative detection reagent box performance evaluation " and openly CRP monoclonal polyclonal antibody EDC/NHS covalent method is combined with carboxylic polystyrene microsphere, prepare the reagent detecting CRP by latex immunoturbidimetry.Protein carrier microballoon is generally made with the macromolecular material of Prof. Du Yucang, has different performances with the microballoon that different monomer polymerizations becomes.Take styrene as monomer by the microballoon that the diameter that emulsion polymerization is made is about 0.8 μm be conventional microsphere supported, the suspension of this kind of microballoon is emulsus, has good suspension property.But this kit detectability 0.72mg/L, only qualitatively near this value can determine to contain CRP to be measured in sample, accurately cannot distinguish the critical value 1mg/L of super quick CRP; According to the regression equation data that article is delivered, the accuracy in conjunction with instrument itself calculates, and the critical point of Accurate Determining sample will be greater than 1mg/L; This reagent is only applicable to special protein instrument in addition, and special protein instrument feature is single inspection, and the used time is short, but non-processor great amount of samples, cannot detect by high flux on automatic clinical chemistry analyzer, therefore fully cannot meet the needs of clinical diagnosis.
Turbidimetry is used for Biochemical Analyzer, and Biochemical Analyzer cardinal principle to use in spectral technique not homoatomic extinction different and measures, and so for the functional realiey of ISE module, mainly contain two kinds of methods, one is colourimetry, and two is indirect methods.Colourimetry is because of its measuring accuracy, and accuracy differs too large with required, and this method uses in the earlier laboratory of medical science checks, has been belong to superseded usage.Indirect method, its Method And Principle is similar to the Other Instruments existed in the market direct method used, but caused by the fragility of ACA, for anti-instrument internal is blocked, very strict to the requirement of sample, need be separated through routine and can measure after dilution again, and general biochemical ISE module to the extension rate of sample mostly at about 30 times, under so large extension rate, useful really to pipeline, but from data statistics processing angle, such measurement, error will be amplified in proportion, the result measured so like this, accuracy and precision can not reach requirement.
Summary of the invention
Technical matters to be solved by this invention is for above-mentioned the deficiencies in the prior art, and provide a kind of mixed size grain size latex and improve emulsion reagent sensitivity and linear method simultaneously, have highly sensitive, the range of linearity is wide, saves the advantages such as antibody consumption.
A kind ofly improve emulsion reagent sensitivity and linear method, detection kit comprises reagent R1 and reagent R2, it is characterized in that, described emulsion reagent is the emulsion reagent of mixed size grain size latex, make c reactive protein latex than turbid detection kit, this kit comprises reagent R1 and reagent R2; Described method comprises the steps:
1) by mouse determined antigen monoclonal antibody orientation bag by the latex microsphere of Large stone,
2) by rabbit or sheep determined antigen polyclonal antibody orientation bag by the latex microsphere of small particle diameter;
3) then by bag by after large grain size latex and small grain size latex mix.
Preferred embodiment is, described determined antigen is c reactive protein;
Preferred embodiment is, described latex microsphere is the polystyrene latex microspheres of carboxyl modified;
The mean grain size of described large grain size latex microballoon is 200-400nm, preferred 300-350;
The mean grain size of described small grain size latex microballoon is 50-200nm, preferred 50-100nm.
Preferred embodiment is, described wrap anti-c reactive protein antibody orientation by antibody used to large grain size latex microballoon is mouse monoclonal antibody, and ELISA tires and is greater than 1:100000; Bag is rabbit polyclonal antibody or sheep polyclonal antibody by antibody used to large grain size latex microballoon, and ELISA tires and is greater than 1:50000.
Described is comprised anti-c reactive protein antibody orientation bag by the preparation process on large grain size latex or small particle diameter microballoon:
(1) activation of polystyrene latex microspheres: add ethyldimethyl amine carbodiimide in the polystyrene latex microspheres of carboxyl, mix, add adipic dihydrazide after reaction terminates, continue reaction, obtain the polystyrene latex microspheres activated;
(2) oxidation of anti-c reactive protein antibody: the hydroxyl oxygen on anti-c reactive protein antibody Fc end sugar chain is changed into aldehyde radical with sodium metaperiodate;
(3) coupling of anti-c reactive protein antibody and polystyrene latex microspheres: the anti-c reactive protein antibody that the polystyrene latex microspheres activate step (1) and step (2) are oxidized mixes and reacts, after reaction terminates, add the group that glucose capped polystyrene latex microsphere does not react, obtain the polystyrene latex microspheres of the anti-c reactive protein antibody of coupling.
The preparation of described reagent R2 comprises:
1), get Large stone or small particle diameter polystyrene latex particles, wash three times with MES buffer soln MES, dispersion;
2), add with MES solution freshly prepared ethyldimethyl amine carbodiimide EDAC solution, room temperature reaction 1 hour;
3), the freshly prepared adipic acid dihydrazide solution of MES solution that adds, 4 DEG C of reactions are spent the night;
4), use MES solution washing twice, phosphate buffer washing once, disperses for subsequent use;
5) get mouse monoclonal antibody, rabbit or sheep Anti-TNF-α liquid solution, respectively, add the sodium periodate solution of phosphate buffered saline, mixed at room temperature 15 minutes;
6), by antibody well-oxygenated in step 5) join in the polystyrene latex microspheres solution activated in step 4), wherein mouse monoclonal antibody is used for bag by large grain size latex microballoon, and rabbit or sheep polyclonal antibody are used for bag by small particle diameter microballoon, and 4 DEG C of reactions are spent the night;
7), add bovine serum albumin solution, 4 DEG C are reacted 2 hours;
8), add 10% glucose solution, 4 DEG C of reactions are spent the night;
9), with ammonium chloride buffer wash three times, add containing 1.5% sucrose, 0.2% Sodium azide, ammonium chloride buffer to latex microsphere final concentration is 0.25%.
10), by the large grain size latex microballoon of preparation and small grain size latex microballoon mix according to the volume ratio of 1:5, complete the preparation of reagent R2.
Further,
According to mass fraction meter,
In step 1), ethyldimethyl amine carbodiimide: polystyrene latex particles is 0.1-1:100, preferred 0.5:100;
In step 3), adipic dihydrazide: polystyrene latex particles is 10-50:100, preferred 50:100.
In step 5), according to mass fraction meter, sodium metaperiodate: antibody is 1:2, wherein, antibody is mouse monoclonal antibody, rabbit or sheep polyclonal antibody.
In step 6), according to mass fraction meter, antibody: the polystyrene latex microspheres of activation is 5-20:100, preferred 10-15:100, in step 8), glucose: the polystyrene latex microspheres of activation is 20-50:100, preferred 30-40:100.
In described mixed size grain size latex microballoon, large grain size latex microballoon: small grain size latex microspheres quality is than being 1:3-10, preferred 2-5, more preferably 2-3.
The formula of described R1 is: Tris50mmol/L, and the concentration of sucrose is 5g/L, and the concentration of Sodium azide is the concentration of 1g/L, PEG8000 is 70g/L, stachyose 1.6-3g/L; Alum 0.6-1g/L; Fructose Diphosphate 0.8-3g/L; Sodium hexametaphosphate 0.09-0.3g/L.
In described reagent 2, in dispersion liquid, the concentration of Tris-HCl damping fluid is 30mM, and the concentration of bovine serum albumin(BSA) BSA is 5g/L, and the concentration of Sodium azide is 1g/L.
The volume ratio of described seminal plasma fructose detection kit R1 and reagent R2 is 3:1.
Described mixed size grain size latex improves emulsion reagent sensitivity and linear method simultaneously, is used for preparing c reactive protein latex than turbid detection kit, and this kit comprises reagent R1 and reagent R2;
The formula of described reagent R1 is: Tris50mmol/L, and the concentration of sucrose is 5g/L, and the concentration of Sodium azide is the concentration of 1g/L, PEG6000 is 70g/L, stachyose 1.6-3g/L; Alum 0.6-1g/L; Fructose Diphosphate 0.8-3g/L; Sodium hexametaphosphate 0.09-0.3g/L.
In described reagent R2, in dispersion liquid, the concentration of Tris-HCl damping fluid is 30mM, and the concentration of bovine serum albumin(BSA) BSA is 5g/L, and the concentration of Sodium azide is 1g/L.
A technical scheme of the present invention is,
1), 1ml(100mg/ml is got) Large stone or small grain size latex microballoon, wash three times with the MES solution (MES damping fluid) of 0.2M, pH5.0, dispersion;
2), 0.05ml 0.2M is added, the MES solution freshly prepared 10mg/ml ethyldimethyl amine carbodiimide EDAC solution of pH5.0, room temperature reaction 1 hour;
3), add 0.5ml 0.2M, the MES solution freshly prepared 100mg/ml adipic acid dihydrazide solution of pH5.0,4 DEG C of reactions are spent the night;
4), with the phosphate buffer of the MES solution washing twice, 0.1M, pH7.5 of 0.2M, pH5.0 wash once, disperse for subsequent use;
5), get 1.5ml mouse monoclonal antibody or rabbit polyclonal antibody (10mg/ml) solution, add 0.75ml 0.1M, the sodium periodate solution of the 10mg/ml of the phosphate buffered saline of pH7.5, mixed at room temperature 15 minutes;
6), by antibody well-oxygenated in step 5 join in the latex microsphere solution activated in step 4, wherein mouse monoclonal antibody is used for bag by large grain size latex microballoon, and rabbit polyclonal antibody is used for bag by small particle diameter microballoon, and 4 degree of reactions are spent the night;
7), add 0.5ml10% bovine serum albumin solution, 4 DEG C are reacted 2 hours;
8), add 0.5ml10% glucose solution, 4 DEG C of reactions are spent the night;
9), with the ammonium chloride buffer of 0.2M, pH7.5 wash three times, add containing 1.5% sucrose, ammonium chloride buffer to the latex microsphere final concentration of the 0.2M of 0.2% Sodium azide, pH7.5 is 0.25%.
10), by the large grain size latex microballoon of preparation and small grain size latex microballoon mix according to the volume ratio of 1:5, complete the preparation of reagent R2.
C reactive protein albumen calibration object: the c reactive protein adding different content in 0.9% physiological saline, aseptic filtration.In described c reactive protein calibration object, c reactive protein content controls between 0-160mg/l, and these calibration objects are colourless transparent liquid.
Another embodiment of the invention is,
Being formulated as follows of the main agents of the present embodiment:
Reagent R1: containing 0.3%PEG8000,3% sodium chloride, 0.5% sucrose, 0.5% polysorbas20, the 0.2M ammonium chloride buffer of 0.2% Sodium azide, this reagent is colourless transparent solution.
Mixed size particle diameter polystyrene latex microspheres containing 0.25% anti-c reactive protein antibody sensitized in reagent R2:0.2M ammonium chloride buffer, 1.5% sucrose, 0.2% Sodium azide.This reagent is milky white solution.
Concrete preparation process is as follows:
1), 1ml(100mg/ml is got) Large stone or small grain size latex microballoon, wash three times with the MES solution (MES damping fluid) of 0.2M, pH5.0, dispersion;
2), 0.1ml 0.2M is added, the MES solution freshly prepared 10mg/ml ethyldimethyl amine carbodiimide EDAC solution of pH5.0, room temperature reaction 1 hour;
3), add 0.1ml 0.2M, the MES solution freshly prepared 100mg/ml adipic acid dihydrazide solution of pH5.0,4 DEG C of reactions are spent the night;
4), with the phosphate buffer of the MES solution washing twice, 0.1M, pH7.5 of 0.2M, pH5.0 wash once, disperse for subsequent use;
5), get 1.5ml mouse monoclonal antibody or rabbit polyclonal antibody (10mg/ml) solution, add 0.75ml 0.1M, the sodium periodate solution of the 10mg/ml of the phosphate buffered saline of pH7.5, mixed at room temperature 15 minutes;
6), by antibody well-oxygenated in step 5 join in the latex microsphere solution activated in step 4, wherein mouse monoclonal antibody is used for bag by large grain size latex microballoon, and rabbit polyclonal antibody is used for bag by small particle diameter microballoon, and 4 degree of reactions are spent the night;
7), add 0.5ml10% bovine serum albumin solution, 4 DEG C are reacted 2 hours;
8), add 0.5ml10% glucose solution, 4 DEG C of reactions are spent the night;
9), with the ammonium chloride buffer of 0.2M, pH7.5 wash three times, add containing 1.5% sucrose, ammonium chloride buffer to the latex microsphere final concentration of the 0.2M of 0.2% Sodium azide, pH7.5 is 0.25%.
10), by the large grain size latex microballoon of preparation and small grain size latex microballoon mix according to the volume ratio of 1:5, complete the preparation of reagent R2.
In mentioned reagent box of the present invention, the volume ratio of reagent R1 and reagent R2 is 3:1.
When there is determined antigen in sample, because the specific binding of antigen-antibody is connected to each other between the above-mentioned large small grain size latex being coated with antibody, turbidity is caused to rise, determined antigen is more, absorbance increase is more, therefore by the increase of working sample absorbance, the content of determined antigen in sample can be calculated according to typical curve, wherein large grain size latex can guarantee the sensitivity of reagent, small grain size latex can guarantee the linear of reagent, can be improved the sensitivity of emulsion reagent with linear by mixed size grain size latex simultaneously.
The present invention compared with prior art, has following advantage:
1, the present invention adopts the method for mixed size grain size latex microballoon, high sensitivity and linearly wide can be obtained simultaneously, wherein large grain size latex can guarantee the sensitivity of reagent, small grain size latex can guarantee the linear of reagent, solve c reactive protein common at present and detect reagent or desensitization, reduce linear technical matters, effectively can ensure the accuracy that c reactive protein project detects at low value and high level place.
2, directed coupling method is adopted in the present invention by anti-c reactive protein antibody bag by polystyrene latex microspheres, orientation is held to be coupled on polystyrene latex microspheres the Fc in the nonbinding active region of anti-c reactive protein antibody, the false rising of the testing result that the interference that rheumatoid factor (RF) and heterophil antibody can not occur causes
3, directed coupling method is adopted in the present invention by anti-c reactive protein antibody bag by polystyrene latex microspheres, its antigen binding site points to mobile phase, antibody coupling position is Fc fragment, therefore the situation that antigen binding capacity reduces can not be there is, maintain antibody vigor, thus greatly reduce antibody consumption, reduce production cost.
4, mixed size grain size latex of the present invention improves emulsion reagent sensitivity and linear method simultaneously and can improve the sensitivity of emulsion reagent and linear simultaneously, and sensing range reaches 0.8-160mg/l.
Accompanying drawing explanation
Fig. 1; From grinding kit and hospital's definite value serum correlation analysis.
Embodiment
Below by embodiment, the present invention is described in further detail, but the present invention is not only confined to following examples.
The preparation of the c reactive protein detection kit of embodiment 1 mixed size grain size latex microballoon
The main raw material(s) that kit prepared by employing mixed size grain size latex microballoon of the present invention relates to is as follows:
1, anti-c reactive protein antibody: mouse monoclonal antibody, ELISA method measures tires as 1:100000; Rabbit polyclonal antibody, ELISA method measures and tires as 1:50000.
2, latex microsphere: the polystyrene latex microspheres of the employing band carboxylic group that the present invention is only exemplary is tested, and wherein the mean grain size of large grain size latex microballoon is 300-350nm, and the mean grain size of small grain size latex microballoon is 50-100nm.
Being formulated as follows of the main agents of the present embodiment:
Reagent R1: containing 0.3%PEG8000,3% sodium chloride, 0.5% sucrose, 0.5% polysorbas20, the 0.2M ammonium chloride buffer of 0.2% Sodium azide, this reagent is colourless transparent solution.
Mixed size particle diameter polystyrene latex microspheres containing 0.25% anti-c reactive protein antibody sensitized in reagent R2:0.2M ammonium chloride buffer, 1.5% sucrose, 0.2% Sodium azide.This reagent is milky white solution.
Concrete preparation process is as follows:
1), 1ml(100mg/ml is got) Large stone or small grain size latex microballoon, wash three times with the MES solution (MES damping fluid) of 0.2M, pH5.0, dispersion;
2), 0.05ml 0.2M is added, the MES solution freshly prepared 10mg/ml ethyldimethyl amine carbodiimide EDAC solution of pH5.0, room temperature reaction 1 hour;
3), add 0.5ml 0.2M, the MES solution freshly prepared 100mg/ml adipic acid dihydrazide solution of pH5.0,4 DEG C of reactions are spent the night;
4), with the phosphate buffer of the MES solution washing twice, 0.1M, pH7.5 of 0.2M, pH5.0 wash once, disperse for subsequent use;
5), get 1.5ml mouse monoclonal antibody or rabbit polyclonal antibody (10mg/ml) solution, add 0.75ml 0.1M, the sodium periodate solution of the 10mg/ml of the phosphate buffered saline of pH7.5, mixed at room temperature 15 minutes;
6), by antibody well-oxygenated in step 5 join in the latex microsphere solution activated in step 4, wherein mouse monoclonal antibody is used for bag by large grain size latex microballoon, and rabbit polyclonal antibody is used for bag by small particle diameter microballoon, and 4 degree of reactions are spent the night;
7), add 0.5ml10% bovine serum albumin solution, 4 DEG C are reacted 2 hours;
8), add 0.5ml10% glucose solution, 4 DEG C of reactions are spent the night;
9), with the ammonium chloride buffer of 0.2M, pH7.5 wash three times, add containing 1.5% sucrose, ammonium chloride buffer to the latex microsphere final concentration of the 0.2M of 0.2% Sodium azide, pH7.5 is 0.25%.
10), by the large grain size latex microballoon of preparation and small grain size latex microballoon mix according to the volume ratio of 1:5, complete the preparation of reagent R2.
C reactive protein albumen calibration object: the c reactive protein adding different content in 0.9% physiological saline, aseptic filtration.In described c reactive protein calibration object, c reactive protein content controls between 0-160mg/l, and these calibration objects are colourless transparent liquid.
Embodiment 2
Being formulated as follows of the main agents of the present embodiment:
Reagent R1: containing 0.3%PEG8000,3% sodium chloride, 0.5% sucrose, 0.5% polysorbas20, the 0.2M ammonium chloride buffer of 0.2% Sodium azide, this reagent is colourless transparent solution.
Mixed size particle diameter polystyrene latex microspheres containing 0.25% anti-c reactive protein antibody sensitized in reagent R2:0.2M ammonium chloride buffer, 1.5% sucrose, 0.2% Sodium azide.This reagent is milky white solution.
Concrete preparation process is as follows:
1), 1ml(100mg/ml is got) Large stone or small grain size latex microballoon, wash three times with the MES solution (MES damping fluid) of 0.2M, pH5.0, dispersion;
2), 0.1ml 0.2M is added, the MES solution freshly prepared 10mg/ml ethyldimethyl amine carbodiimide EDAC solution of pH5.0, room temperature reaction 1 hour;
3), add 0.1ml 0.2M, the MES solution freshly prepared 100mg/ml adipic acid dihydrazide solution of pH5.0,4 DEG C of reactions are spent the night;
4), with the phosphate buffer of the MES solution washing twice, 0.1M, pH7.5 of 0.2M, pH5.0 wash once, disperse for subsequent use;
5), get 1.5ml mouse monoclonal antibody or rabbit polyclonal antibody (10mg/ml) solution, add 0.75ml 0.1M, the sodium periodate solution of the 10mg/ml of the phosphate buffered saline of pH7.5, mixed at room temperature 15 minutes;
6), by antibody well-oxygenated in step 5 join in the latex microsphere solution activated in step 4, wherein mouse monoclonal antibody is used for bag by large grain size latex microballoon, and rabbit polyclonal antibody is used for bag by small particle diameter microballoon, and 4 degree of reactions are spent the night;
7), add 0.5ml10% bovine serum albumin solution, 4 DEG C are reacted 2 hours;
8), add 0.5ml10% glucose solution, 4 DEG C of reactions are spent the night;
9), with the ammonium chloride buffer of 0.2M, pH7.5 wash three times, add containing 1.5% sucrose, ammonium chloride buffer to the latex microsphere final concentration of the 0.2M of 0.2% Sodium azide, pH7.5 is 0.25%.
10), by the large grain size latex microballoon of preparation and small grain size latex microballoon mix according to the volume ratio of 1:5, complete the preparation of reagent R2.
The property indices test of the c reactive protein detection kit of test example 1 mixed size grain size latex microballoon
1, linear determination test
The sample of the different c reactive protein content of Example 1 sample determination 10, each test sample 3 times, averaged, judges according to judgment basis r2>=0.990.The sensing range of the c reactive protein detection kit that result display adopts mixed size grain size latex micro-sphere method of the present invention to prepare can reach 0.8-160mg/l(in table 1)
Table 1
2, accuracy test
In the human serum sample removing c reactive protein, add the standard solution of certain volume, obtain the c reactive protein sample of 5 different contents.Each sample measures three times, obtains mean value, calculates the recovery.Result show c reactive protein detection kit prepared by mixed size grain size latex micro-sphere method of the present invention swing in the recovery of each concentration point be all less than 5%(in table 2).
Table 2
3, correlation analysis
Obtain with conventional c reactive protein latex intensified than turbid detection kit and the hs-CRP latex intensified serum 200 parts than turbid detection kit definite value from clinical, measure by c reactive protein detection kit prepared by mixed size grain size latex micro-sphere method of the present invention, measurement result and hospital's definite value result are analyzed, result display sample mean deviate is all less than 10%, maximum deviation is less than 15%, R2>0.98, consistency coefficient >0.95, show that the method that the present invention adopts measures accurately, effectively can improve sensitivity, increase the range of linearity.
Above-mentioned detailed description is illustrating for one of them possible embodiments of the present invention, and this embodiment is also not used to limit the scope of the claims of the present invention, and the equivalence that all the present invention of disengaging do is implemented or changed, and all should be contained in the scope of technical solution of the present invention.
Claims (10)
1. one kind is improved emulsion reagent sensitivity and linear method, its detection kit used comprises reagent R1 and reagent R2, it is characterized in that, the emulsion reagent of particle diameter mixing latex sized by described emulsion reagent, make c reactive protein latex than turbid detection kit, this kit comprises reagent R1 and reagent R2; Described method comprises the steps:
1) by mouse determined antigen monoclonal antibody orientation bag by the latex microsphere of Large stone,
2) by rabbit or sheep determined antigen polyclonal antibody orientation bag by the latex microsphere of small particle diameter;
3) then by bag by after large grain size latex and small grain size latex mix.
2. raising emulsion reagent sensitivity as claimed in claim 1 and linear method, is characterized in that,
Described determined antigen is c reactive protein;
Described latex microsphere is the polystyrene latex microspheres of carboxyl modified;
The mean grain size of described large grain size latex microballoon is 200-400nm, preferred 300-350;
The mean grain size of described small grain size latex microballoon is 50-200nm, preferred 50-100nm.
3. raising emulsion reagent sensitivity as claimed in claim 1 and linear method, is characterized in that, described wrap anti-c reactive protein antibody orientation by antibody used to large grain size latex microballoon is mouse monoclonal antibody, and ELISA tires and is greater than 1:100000; Bag is rabbit polyclonal antibody or sheep polyclonal antibody by antibody used to large grain size latex microballoon, and ELISA tires and is greater than 1:50000.
4. raising emulsion reagent sensitivity as claimed in claim 1 and linear method, is characterized in that,
The preparation process of described reagent R2 comprises:
1), get Large stone or small particle diameter polystyrene latex particles, wash three times with MES buffer soln MES, dispersion;
2), add with MES solution freshly prepared ethyldimethyl amine carbodiimide EDAC solution, room temperature reaction 1 hour;
3), the freshly prepared adipic acid dihydrazide solution of MES solution that adds, 4 DEG C of reactions are spent the night;
4), use MES solution washing twice, phosphate buffer washing once, disperses for subsequent use;
5) get mouse monoclonal antibody, rabbit or sheep Anti-TNF-α liquid solution, respectively, add the sodium periodate solution of phosphate buffered saline, mixed at room temperature 15 minutes;
6), by antibody well-oxygenated in step 5) join in the polystyrene latex microspheres solution activated in step 4), wherein mouse monoclonal antibody is used for bag by large grain size latex microballoon, and rabbit or sheep polyclonal antibody are used for bag by small particle diameter microballoon, and 4 DEG C of reactions are spent the night;
7), add bovine serum albumin solution, 4 DEG C are reacted 2 hours;
8), add 10% glucose solution, 4 DEG C of reactions are spent the night;
9), with ammonium chloride buffer wash three times, add containing 1.5% sucrose, 0.2% Sodium azide, ammonium chloride buffer to latex microsphere final concentration is 0.25%;
10), by the large grain size latex microballoon of preparation and small grain size latex microballoon mix according to the volume ratio of 1:5, complete the preparation of reagent R2.
5. raising emulsion reagent sensitivity as claimed in claim 1 and linear method, is characterized in that,
According to mass fraction meter,
In step 1), ethyldimethyl amine carbodiimide: polystyrene latex particles is 0.1-1:100, preferred 0.5:100;
In step 3), adipic dihydrazide: polystyrene latex particles is 10-50:100, preferred 50:100.
6. raising emulsion reagent sensitivity as claimed in claim 1 and linear method, is characterized in that,
In step 5), according to mass fraction meter, sodium metaperiodate: antibody is 1:2, wherein, antibody is mouse monoclonal antibody, rabbit or sheep polyclonal antibody.
7. raising emulsion reagent sensitivity as claimed in claim 1 and linear method, is characterized in that,
In step 6), according to mass fraction meter, antibody: the polystyrene latex microspheres of activation is 5-20:100, preferred 10-15:100, in step 8), glucose: the polystyrene latex microspheres of activation is 20-50:100, preferred 30-40:100.
8. raising emulsion reagent sensitivity as claimed in claim 1 and linear method, is characterized in that,
In described mixed size grain size latex microballoon, large grain size latex microballoon: small grain size latex microspheres quality is than being 1:3-10, preferred 2-5, more preferably 2-3.
9. raising emulsion reagent sensitivity as claimed in claim 1 and linear method, is characterized in that,
The formula of described R1 is: Tris50mmol/L, and the concentration of sucrose is 5g/L, and the concentration of Sodium azide is the concentration of 1g/L, PEG8000 is 70g/L, stachyose 1.6-3g/L; Alum 0.6-1g/L; Fructose Diphosphate 0.8-3g/L; Sodium hexametaphosphate 0.09-0.3g/L;
In described reagent 2, in dispersion liquid, the concentration of Tris-HCl damping fluid is 30mM, and the concentration of bovine serum albumin(BSA) BSA is 5g/L, and the concentration of Sodium azide is 1g/L.
10. raising emulsion reagent sensitivity as claimed in claim 9 and linear method, is characterized in that,
The volume ratio of described seminal plasma fructose detection kit R1 and reagent R2 is 3:1.
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