CN104049085B - A kind of CRP latex enhancing immune turbidimetric assay reagent and kit thereof - Google Patents
A kind of CRP latex enhancing immune turbidimetric assay reagent and kit thereof Download PDFInfo
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Abstract
The invention provides a kind of CRP latex enhancing immune turbidimetric assay reagent, described CRP latex enhancing immune turbidimetric assay reagent comprises the latex particle of CRP antibody labeling, damping fluid, surfactant, inorganic salts, stabilizing agent, suspending agent, excipient and antiseptic.The present invention also provides a kind of single reagent CRP latex enhancing immune turbidimetric assay kit and application thereof.Reagent composition of the present invention is simple, simple to operation, and fast, reagent stability is good in reaction, highly sensitive, reproducible, use instrument simple, adapt to instrument extensive, with low cost, can be widely used for large, middle and small hospital, outpatient clinic quick diagnosis infects and infects the evaluation of prognosis.
Description
Technical field
The present invention relates to medical science, be specifically related to a kind of CRP latex enhancing immune than turbid quantitative measurement reagent and kit thereof and application thereof.
Background technology
CRP is one of albumen important in Acute reaction protein, is a kind of marker of inflammation of sensitivity, and its blood concentration sharply raises at acute injury and when infecting.In normal human serum, CRP content is very micro-, but in the situations such as infection, inflammatory disease, tissue damage, operation wound and necrosis, raises rapidly in several hours, and continues sharply to rise, and can reach peak at 24 ~ 72h.After pathology disappears, CRP can be dropped rapidly to normally.The order of severity of CRP ascending velocity, amplitude and duration and the state of an illness and tissue damage is closely related, and not by the impact of the treatment meanss such as radiotherapy, chemotherapy, cortin.Therefore, compare with other acute phase proteins with traditional inspection item, CRP is more reliably sensitive.
Latex enhancing immune turbidimetry (particle-enhancedturbidimetricimmune-assay, PETIA) be that the one that occurs in recent years is comparatively stable, body fluid albumen homogeneous phase immunoturbidimetry assay method accurately, be widely used in clinical diagnosis field at present.PETIA method is the surface-crosslinked antibody at polymer latex microballoon, when crosslinked have the microballoon of antibody to be combined with antigen after, can flock together rapidly at short notice, change astigmatism performance or the light transmission of reactant liquor.The change of reactant liquor astigmatism performance or light transmission (namely inhaling brightness) and the concentration of tested antigen have inherent correlativity, can reflect the concentration of tested antigen within the specific limits.CRP latex enhancing immune than turbid be exactly the prominent example of this kind of assay method, it is the mensuration of carrying out antigen, antibody response and result in homogeneous reaction system.
CRP latex enhancing immune turbidimetric assay reactant liquor commercially available at present and kit are double reagent, by reaction buffer 1(R1), the latex intensified particle reagents 2(R2 of CRP antibody labeling) composition.R1, R2 mix in advance and easily cause antibody inactivation, latex particle in R2 easily to assemble, and kit can not be preserved for a long time.Especially for the mensuration of whole blood sample, current commercial reagent box needs first haemolysis, rear mensuration two step to operate, and both expended time in, repeatability is also bad.
Summary of the invention
The object of this invention is to provide a kind of simple and quick, easy preservation, reproducible can the single reagent CRP latex enhancing immune of quantitative measurement CRP than turbid quantitative measurement reagent and kit thereof.
For achieving the above object, technical scheme of the present invention is: a kind of CRP latex enhancing immune turbidimetric assay reagent, and described CRP latex enhancing immune turbidimetric assay reagent comprises the latex particle of CRP antibody labeling, damping fluid, surfactant, inorganic salts, stabilizing agent, suspending agent, excipient and antiseptic.
Preferably, the diameter of described latex particle is 50nm-4000nm; Its finishing group is one in-COOH ,-NH2 ,-SH ,-OH ,-CHO or physisorption type latex particle.
Preferably, the diameter of described latex particle is 100-250nm and finishing group is the one in-COOH or-NH2.
Preferably, described stabilizing agent is the one in bovine serum albumin(BSA), gelatin or casein; Described inorganic salts are a kind of, two or more combination in sodium chloride, potassium chloride, lime chloride, ammonium chloride, sodium sulphate, potassium sulfate; Described surfactant is a kind of, two or more combination in polysorbas20, polysorbate40, Tween 80, triton x-100, span 40, sorbester p18, saponin, cetyl-3-ammonio methacrylate, hexadecylamino ammonium bromide, lysophosphatide; Described excipient is a kind of, two or more combination in sucrose, glucose, maltose, trehalose, sweet mellow wine; Described suspending agent is a kind of, two or more combination in ethylene glycol, glycerine, lactose; Described antiseptic is the one in Sodium azide, thimerosal, Proclin-300.
Preferably, described surfactant is the one that existing dissolving haemocyte function has again in the reagent triton x-100 of surfactant function, Tween-20.
Preferably, described excipient is the trehalose of concentration 1%-5%.
Preferably, described suspending agent is the glycerine of concentration 1%-20%.
Preferably, the regulating power of described damping fluid requires that pH scope is between 7.0-9.0, and concentration range is 10-100mmol/L.
Preferably, described damping fluid is 3-[N, N-bis-(hydroxyethyl) is amino]-2-hydroxy-propanesulfonic acid-sodium hydrate buffer solution (DIPSO-NaOH), 4-(2-hydroxyethyl)-1-piperazine propane sulfonic acid-sodium hydrate buffer solution (HEPPS-NaOH), 3-(N-morpholinyl) propane sulfonic acid-sodium hydrate buffer solution (MOPS-NaOH), N-(2-hydroxyethyl) piperazine-N'-2-ethane sulfonic acid-NaOH damping fluid (HEPES-NaOH), trishydroxymethylaminomethane-HCL damping fluid (Tris-HCL), phosphate buffer, imidazole buffer, glycine-NaOH buffer, one in barbitol buffer solution.
Preferably, described antiseptic is Sodium azide, and concentration is 0.1%.
Quick haemolysis function when the stability that in single reagent CRP latex enhancing immune turbidimetric assay reagent, reagent stores and mensuration whole blood sample is extremely important.We select plurality of reagents to combine according to the principle of stabilized latex particle, rapid solution haemocyte, and wherein following reagent is best of breed:
In described kit, the regulating power of damping fluid requires that pH scope is between 7.0-9.0, preferred DIPSO-NaOH damping fluid.
Described DIPSO-NaOH pH of buffer is between 6.8-8.2, and concentration range is 10-100mmol/L, is preferably 30-60mmol/L.
Surfactant has multiple choices, and preferred existing dissolving haemocyte function has again reagent triton x-100 or the Tween-20 of surfactant function.They have rapid solution red blood cell and leukocytic function, can be adsorbed onto again the function on latex particle surface, make the unsuitable aggregate and precipitate of latex particle, thus have ensured that latex particle can long-term preservation in a liquid state.Preferred triton x-100, concentration is 0.1%-0.5%.
Inorganic salts can maintain the osmotic pressure of solution, and the long-term stability being conducive to antibody activity is preserved, and preferred NaCL, concentration is 0.6%-1%.
Stabilizing agent, can play good colloidal dispersions and bioactive protective effect to the antibody of latex particle surface indicia.Preferred BSA, concentration is 0.5%-2%.
Suspending agent, latex particle can well be made to disperse, be suspended in solution, and be conducive to the colloidization of latex particle, long-term preservation does not precipitate.Preferred glycerine, concentration is 1%-10%.
Excipient, the long-term stability being conducive to antibody is preserved, and preferred trehalose, concentration is 1%-5%.
Antiseptic, preferred Sodium azide, concentration is 0.1%.
During mensuration, the antigen-antibody reaction that in sample, CRP and CRP antibody latex particle occurs makes the turbidity of reaction system rise, and the transmittance in reaction system can be caused to reduce, and the CRP content in its rate of change and sample is inversely proportional to.Scattered light strengthens, the CRP content direct ratio in its rate of change and sample.Adopt transilluminator or scattering turbidimetry instrument to measure the rate of change of transmitted light or scattered light, with reference to the calibration curve set up by CRP calibration object concentration, thus the content of CRP can be calculated.
The present invention also provides a kind of single reagent CRP latex enhancing immune turbidimetric assay kit, described single reagent CRP latex enhancing immune turbidimetric assay kit comprises at least one detector tube, is loaded with CRP latex enhancing immune turbidimetric assay reagent of the present invention in detector tube.
The present invention also provides a kind of application of single reagent CRP latex enhancing immune turbidimetric assay kit, and described single reagent CRP latex enhancing immune turbidimetric assay kit is applicable to various transilluminator or scatterometer.
Concentration described in the present invention, as do not carried out specified otherwise, is the grams of contained solute in quality concentration of volume percent or 100ml solution.
The present invention compared with prior art, has following features:
1. reagent composition is simple, only has single agents.
2. simple to operate, only sample directly need be added in single reagent CRP reactant liquor, without the need to
First reagent adding 1 as commercially available prod, then add sample, then reagent adding 2, complex operation, time-consumingly takes a lot of work.
3., for whole blood sample, single reagent CRP reactant liquor can haemolysis, can detect again in sample
CRP content.
4. fast, whole reaction only needs 60-90 second in reaction.
5. reagent stability is good, can preserve 18 months for 4 degrees Celsius, can preserve 15 days after uncork
There is not obvious change in quality.
6. adapt to instrument extensive, transilluminator, scatterometer all can mate.Be made into
POCT reagent more meets the demand of outpatient service, emergency treatment.
Accompanying drawing explanation
Fig. 1 is the calibration curve of the embodiment of the present invention 1 in biochemical instruments.
Fig. 2 is the range of linearity correlativity of the embodiment of the present invention 1 in biochemical instruments.
Fig. 3 is the calibration curve of the embodiment of the present invention 2 on scattering turbidimetry instrument
Fig. 4 is the range of linearity correlativity of the embodiment of the present invention 2 on scattering turbidimetry instrument.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read content of the present invention, these equivalent form of values fall within equally the application the scope that limits of attached claims.
Embodiment 1
One, the preparation of reagent and kit is measured:
1. the polystyrene latex particles of surface carboxyl groups (103nm) the MES damping fluid of the pH6.0 of 50mM is diluted to 10ml, final concentration is 1%;
2. add EDC200 milligram in above-mentioned solution, add the CRP antibody of 10 milligrams of affinity chromatographys, mixed at room temperature 2 hours;
3. the glycocoll adding 10ml100mM reacts 2 hours in above-mentioned solution, closes the active group of latex particle excess surface;
Centrifugal 20 minutes of 4.12000rpm, removes supernatant;
5. with following buffer, the CRP antibody latex particle of above-mentioned mark is diluted to working concentration, is equipped with calibration object and is single reagent CRP latex enhancing immune than turbid kit.Single reagent is filled a prescription: 35mMDIPSO-NaOH, pH7.0; 0.2% triton x-100; 0.7%NaCl; 0.5%BSA; 2% glycerine; 1% trehalose; 0.1% Sodium azide.
6.CRP calibration object: commercially available CRP sterling is dissolved in 50mMTris-HCL, pH7.5,0.85%NaCl, 0.5%BSA, 0.1% Sodium azide, CRP concentration is respectively 0mg/L, 5mg/L, 15mg/L, 50mg/L, 100mg/L, 150mg/L.
Two, the application of kit in biochemical instruments:
1., with 6 variable concentrations CRP calibration object 150mg/L, 100mg/L, 50mg/L, 15mg/L, 5mg/L, 0mg/L coordinate single reagent to calibrate.With wavelength 575nm for measuring wavelength, add reagent R in detector tube, 300ul, 37 degree of reactions 90 seconds. the calibration object 4ul getting variable concentrations respectively adds in above-mentioned detector tube and mixes, the assaying reaction suction brightness value (A1, A2) of the 10th second, the 90th second, calculates the difference DELTA A=A2-A1 inhaling brightness.Using the suction luminance difference Δ A of each calibration tube as ordinate, corresponding concentration, as horizontal ordinate, makes " concentration-absorbance difference " calibration curve.Fig. 1 is the calibration curve of embodiment 1 in biochemical instruments.
2. be measured in the same method the suction luminance difference of blood serum sample (if mensuration whole blood sample need add the sample size of 10ul, plasma sample amount is 6ul), substitute into calibration curve and calculate sample CRP concentration.
3. range of linearity correlativity
By CRP enriched sample (200mg/L), 150.00mg/L is become with normal saline dilution, 50.00mg/L, 25.0mg/L, 10.0mg/L, 5.0mg/L, biochemical instruments measures with the wavelength of 575nm, each concentration replication 3 times, the mean value and theoretical concentration that measure concentration are carried out linear regression analysis, and related coefficient is R
2=0.9979, result: within the scope of 5.0-150mg/L linear determination, range of linearity good relationship (see figure 2).
4. sensitivity (lowest detectable limit) is with 5% human serum albumins for dummy, and replication 20 times, result of calculation average X is 0.05mg/L, standard deviation SD is 0.15.Calculating X+2SD is 0.35mg/L, and therefore this kit measures CRP sensitivity and can reach 0.35mg/L in biochemical instruments.
5. repeatability assessment:
Adopt same batch of reagent to carry out 10 replications to RandoxSpecificProteinControl1 (target value is 24.0mg/L), calculate coefficient of variation CV(< 6%), measurement result is in table 1.
SD=
CV=SD/
×100%
Table 1 repeated assess and determine result table
6. difference between batch assessment: carry out RandoxSpecificProteinControl1 (target value is 24.0mg/L) to three batches of reagent and measure, often criticize reagent and measure 3 times, measurement result gathers in table 2.
Table 2 difference between batch assess and determine result table
embodiment 2
One, the preparation of reagent and kit is measured:
1. the polystyrene latex particles of surface carboxyl groups (150nm) the MES damping fluid of the pH6.0 of 50mM is diluted to 10ml, final concentration is 1%;
2. add EDC200 milligram in above-mentioned solution, add the CRP antibody of 10 milligrams of affinity chromatographys, mixed at room temperature 2 hours;
3. the glycocoll adding 10ml100mM reacts 2 hours in above-mentioned solution, closes the active group of latex particle excess surface;
Centrifugal 20 minutes of 4.12000rpm, removes supernatant;
5. with following buffer, the CRP antibody latex particle of above-mentioned mark is diluted to work dense
Degree, is equipped with calibration object and is single reagent CRP latex enhancing immune than turbid kit.Single reagent is filled a prescription: 45mMMOPS-NaOH, pH7.5; 0.3% polysorbas20; 0.8%NaCl; 1% gelatin; 5% glycerine; 3% trehalose; 0.1% Sodium azide.
6.CRP calibration object: commercially available CRP sterling is dissolved in 50mMTris-HCL, pH7.5,
0.85%NaCl, 0.5%BSA, 0.1% Sodium azide, CRP concentration is respectively 0mg/L, 5mg/L, 15mg/L, 50mg/L, 100mg/L, 150mg/L.
Two, the application of kit on scattering turbidimetry instrument
1., with 6 variable concentrations CRP calibration object 150mg/L, 100mg/L, 50mg/L, 15mg/L, 5mg/L, 0mg/L coordinate single reagent to calibrate.With wavelength 630nm for measuring wavelength, reagent R is added in detector tube, 300ul, 37 degree of reactions 90 seconds. the calibration object 4ul getting variable concentrations respectively adds in above-mentioned detector tube and mixes, the assaying reaction scattered light signal value (A1 of the 10th second, the 90th second, A2), the difference DELTA A=A2-A1 of scattered light signal is calculated.Using the scattered light signal difference DELTA A of each calibration tube as ordinate, corresponding concentration, as horizontal ordinate, makes " concentration-scattered light difference " calibration curve.(see figure 3)
2., if be measured in the same method whole blood sample 10ul(to measure blood serum sample and need add the sample size of 4ul, plasma sample amount is 6ul) scattered light signal difference, substitute into calibration curve and calculate sample CRP concentration.
3. range of linearity correlativity
By CRP enriched sample (180mg/L), 150.00mg/L is become with normal saline dilution, 75.00mg/L, 25.0mg/L, 10.0mg/L, 5.0mg/L, scatterometer measures with the wavelength of 630nm, each concentration replication 3 times, the mean value and theoretical concentration that measure concentration are carried out linear regression analysis, and related coefficient is R
2=0.9969, result: within the scope of 5.0-150mg/L linear determination, range of linearity good relationship (see figure 4).
4. sensitivity (lowest detectable limit)
With 5% human serum albumins for dummy, replication 20 times, result of calculation average X is 0.1mg/L, standard deviation SD is 0.20.Calculating X+2SD is 0.5mg/L, and therefore this kit measures CRP sensitivity and can reach 0.5mg/L on scatterometer.
5. repeatability assessment:
Adopt same batch of reagent to carry out 10 replications to RandoxSpecificProteinControl1 (target value is 24.0mg/L), calculate coefficient of variation CV(< 6%), the results are shown in Table 3.
The repeated assessment result table of table 3
6. difference between batch assessment: carry out RandoxSpecificProteinControl1 (target value is 24.0mg/L) to three batches of reagent and measure, often criticize reagent and measure 3 times, measurement result is in table 4.
Table 4 difference between batch assessment result table
Embodiment 3
One, the preparation of reagent and kit is measured:
1. the polystyrene latex particles of surface amination (200nm) the MES damping fluid of the pH6.0 of 50mM is diluted to 10ml, final concentration is 1%;
2. add EDC200 milligram in above-mentioned solution, add the CRP antibody of 10 milligrams of affinity chromatographys, mixed at room temperature 2 hours;
3. the glycocoll adding 10ml100mM reacts 2 hours in above-mentioned solution, closes latex
The active group that particle surface is unnecessary;
Centrifugal 20 minutes of 4.12000rpm, removes supernatant;
5. with following buffer, the CRP antibody latex particle of above-mentioned mark is diluted to work dense
Degree, is equipped with calibration object and is single reagent CRP latex enhancing immune than turbid kit.Single reagent is filled a prescription: 55mMTris-HCL, pH8.0; 0.4% triton x-100; 0.9%KCl; 1.5%BSA; 8% glycerine; 5% trehalose; 0.1% Sodium azide.
6.CRP calibration object: commercially available CRP sterling is dissolved in 50mMTris-HCL, pH7.5,
0.85%NaCl, 0.5%BSA, 0.1% Sodium azide, CRP concentration is respectively 0mg/L, 5mg/L, 15mg/L, 50mg/L, 100mg/L, 150mg/L.
Two, the application of kit on scattering turbidimetry instrument
1., with 6 variable concentrations CRP calibration object 150mg/L, 100mg/L, 50mg/L, 15mg/L, 5mg/L, 0mg/L coordinate single reagent to calibrate.With wavelength 630nm for measuring wavelength, reagent R is added in detector tube, 300ul, 37 degree of reactions 90 seconds. the calibration object 4ul getting variable concentrations respectively adds in above-mentioned detector tube and mixes, the assaying reaction scattered light signal value (A1 of the 10th second, the 90th second, A2), the difference DELTA A=A2-A1 of scattered light signal is calculated.Using the scattered light signal difference DELTA A of each calibration tube as ordinate, corresponding concentration, as horizontal ordinate, makes " concentration-scattered light difference " calibration curve.
2., if be measured in the same method whole blood sample 10ul(to measure blood serum sample and need add the sample size of 4ul, plasma sample amount is 6ul) scattered light signal difference, substitute into calibration curve and calculate sample CRP concentration.
3. range of linearity correlativity
By CRP enriched sample (180mg/L), 150.00mg/L is become with normal saline dilution, 75.00mg/L, 25.0mg/L, 10.0mg/L, 5.0mg/L, scatterometer measures with the wavelength of 630nm, each concentration replication 3 times, the mean value and theoretical concentration that measure concentration are carried out linear regression analysis, and related coefficient is R
2=0.9945, result: within the scope of 5.0-150mg/L linear determination, range of linearity good relationship.
4. sensitivity (lowest detectable limit)
With 5% human serum albumins for dummy, replication 20 times, result of calculation average X is 0.16mg/L, standard deviation SD is 0.15.Calculating X+2SD is 0.46mg/L, and therefore this kit measures CRP sensitivity and can reach 0.46mg/L on scatterometer.
5. repeatability assessment:
Adopt same batch of reagent to carry out 10 replications to RandoxSpecificProteinControl1 (target value is 24.0mg/L), calculate coefficient of variation CV(< 6%), measurement result is in table 5.
The repeated assessment result table of table 5
6. difference between batch assessment: carry out RandoxSpecificProteinControl1 (target value is 24.0mg/L) to three batches of reagent and measure, often criticize reagent and measure 3 times, measurement result is in table 6.
Table 6 difference between batch assessment result table
Embodiment 4
One, the preparation of reagent and kit is measured:
1. the polystyrene latex particles of surface amination (135nm) the MES damping fluid of the pH6.0 of 50mM is diluted to 10ml, final concentration is 1%;
2. add EDC200 milligram in above-mentioned solution, add the CRP antibody of 10 milligrams of affinity chromatographys, mixed at room temperature 2 hours;
3. the glycocoll adding 10ml100mM reacts 2 hours in above-mentioned solution, closes latex
The active group that particle surface is unnecessary;
Centrifugal 20 minutes of 4.12000rpm, removes supernatant;
5. with following buffer, the CRP antibody latex particle of above-mentioned mark is diluted to work dense
Degree, is equipped with calibration object and is single reagent CRP latex enhancing immune than turbid kit.Single reagent is filled a prescription: 40mMHEPES-NaOH, pH8.0; 0.35% polysorbas20; 0.85%CaCl2; 2% gelatin; 8% glycerine; 5% trehalose; 0.1% Sodium azide.
6.CRP calibration object: commercially available CRP sterling is dissolved in 50mMTris-HCL, pH7.5,
0.85%NaCl, 0.5%BSA, 0.1% Sodium azide, CRP concentration is respectively 0mg/L, 5mg/L, 15mg/L, 50mg/L, 100mg/L, 150mg/L.
Two, the application of kit on scattering turbidimetry instrument
1., with 6 variable concentrations CRP calibration object 150mg/L, 100mg/L, 50mg/L, 15mg/L, 5mg/L, 0mg/L coordinate single reagent to calibrate.With wavelength 630nm for measuring wavelength, reagent R is added in detector tube, 300ul, 37 degree of reactions 90 seconds. the calibration object 4ul getting variable concentrations respectively adds in above-mentioned detector tube and mixes, the assaying reaction scattered light signal value (A1 of the 10th second, the 90th second, A2), the difference DELTA A=A2-A1 of scattered light signal is calculated.Using the scattered light signal difference DELTA A of each calibration tube as ordinate, corresponding concentration, as horizontal ordinate, makes " concentration-scattered light difference " calibration curve.
2., if be measured in the same method whole blood sample 10ul(to measure blood serum sample and need add the sample size of 4ul, plasma sample amount is 6ul) scattered light signal difference, substitute into calibration curve and calculate sample CRP concentration.
3. range of linearity correlativity
By CRP enriched sample (180mg/L), 150.00mg/L is become with normal saline dilution, 75.00mg/L, 25.0mg/L, 10.0mg/L, 5.0mg/L, scatterometer measures with the wavelength of 630nm, each concentration replication 3 times, the mean value and theoretical concentration that measure concentration are carried out linear regression analysis, and related coefficient is R
2=0.9959, result: within the scope of 5.0-150mg/L linear determination, range of linearity good relationship.
4. sensitivity (lowest detectable limit)
With 5% human serum albumins for dummy, replication 20 times, result of calculation average X is 0.10mg/L, standard deviation SD is 0.13.Calculating X+2SD is 0.36mg/L, and therefore this kit measures CRP sensitivity and can reach 0.36mg/L on scatterometer.
5. repeatability assessment:
Adopt same batch of reagent to carry out 10 replications to RandoxSpecificProteinControl1 (target value is 24.0mg/L), calculate coefficient of variation CV(< 6%), measurement result is in table 7.
The repeated assessment result table of table 7
6. difference between batch assessment: carry out RandoxSpecificProteinControl1 (target value is 24.0mg/L) to three batches of reagent and measure, often criticize reagent and measure 3 times, measurement result is in table 8.
Table 8 difference between batch assessment result table
Embodiment 5
One, the preparation of reagent and kit is measured:
1. the polystyrene latex particles of surface amination (245nm) the MES damping fluid of the pH6.0 of 50mM is diluted to 10ml, final concentration is 1%;
2. add EDC200 milligram in above-mentioned solution, add the CRP antibody of 10 milligrams of affinity chromatographys, mixed at room temperature 2 hours;
3. the glycocoll adding 10ml100mM reacts 2 hours in above-mentioned solution, closes latex
The active group that particle surface is unnecessary;
Centrifugal 20 minutes of 4.12000rpm, removes supernatant;
5. with following buffer, the CRP antibody latex particle of above-mentioned mark is diluted to work dense
Degree, is equipped with calibration object and is single reagent CRP latex enhancing immune than turbid kit.Single reagent is filled a prescription: 50mM glycocoll-NaOH, pH7.5; 0.35% triton x-100; 1%KCl; 1% casein; 7% glycerine; 4% trehalose; 0.1% Sodium azide.
6.CRP calibration object: commercially available CRP sterling is dissolved in 50mMTris-HCL, pH7.5,
0.85%NaCl, 0.5%BSA, 0.1% Sodium azide, CRP concentration is respectively 0mg/L, 5mg/L, 15mg/L, 50mg/L, 100mg/L, 150mg/L.
Two, the application of kit on scattering turbidimetry instrument
1., with 6 variable concentrations CRP calibration object 150mg/L, 100mg/L, 50mg/L, 15mg/L, 5mg/L, 0mg/L coordinate single reagent to calibrate.With wavelength 630nm for measuring wavelength, reagent R is added in detector tube, 300ul, 37 degree of reactions 90 seconds. the calibration object 4ul getting variable concentrations respectively adds in above-mentioned detector tube and mixes, the assaying reaction scattered light signal value (A1 of the 10th second, the 90th second, A2), the difference DELTA A=A2-A1 of scattered light signal is calculated.Using the scattered light signal difference DELTA A of each calibration tube as ordinate, corresponding concentration, as horizontal ordinate, makes " concentration-scattered light difference " calibration curve.
2., if be measured in the same method whole blood sample 10ul(to measure blood serum sample and need add the sample size of 4ul, plasma sample amount is 6ul) scattered light signal difference, substitute into calibration curve and calculate sample CRP concentration.
3. range of linearity correlativity
By CRP enriched sample (180mg/L), 150.00mg/L is become with normal saline dilution, 75.00mg/L, 25.0mg/L, 10.0mg/L, 5.0mg/L, scatterometer measures with the wavelength of 630nm, each concentration replication 3 times, the mean value and theoretical concentration that measure concentration are carried out linear regression analysis, and related coefficient is R
2=0.9973, result: within the scope of 5.0-150mg/L linear determination, range of linearity good relationship.
4. sensitivity (lowest detectable limit)
With 5% human serum albumins for dummy, replication 20 times, result of calculation average X is 0.16mg/L, standard deviation SD is 0.20.Calculating X+2SD is 0.56mg/L, and therefore this kit measures CRP sensitivity and can reach 0.56mg/L on scatterometer.
5. repeatability assessment:
Adopt same batch of reagent to carry out 10 replications to RandoxSpecificProteinControl1 (target value is 24.0mg/L), calculate coefficient of variation CV(< 6%), measurement result is in table 9.
The repeated assessment result table of table 9
6. difference between batch assessment: carry out RandoxSpecificProteinControl1 (target value is 24.0mg/L) to three batches of reagent and measure, often criticize reagent and measure 3 times, measurement result is in table 10.
Table 10 difference between batch assess and determine result
The invention provides a kind of CRP latex enhancing immune turbidimetric assay reagent, a kind of single reagent CRP latex enhancing immune turbidimetric assay kit and application thereof, reagent composition of the present invention is simple, simple to operation, and reaction fast, reagent stability is good, highly sensitive, reproducible, use instrument simple, adapt to instrument extensive, with low cost, can be widely used for large, middle and small hospital, outpatient clinic quick diagnosis infects and infects the evaluation of prognosis.
Foregoing is exemplifying of specific embodiments of the invention, for the reagent, equipment, method of operating etc. of wherein not detailed description, should be understood to take this area existing common and conventional reagent, equipment, method of operating etc. to be implemented.
Be more than the description to the embodiment of the present invention, by the above-mentioned explanation to the disclosed embodiments, professional and technical personnel in the field realized or uses the present invention.To be apparent for those skilled in the art to the multiple amendment of these embodiments, General Principle as defined herein can without departing from the spirit or scope of the present invention, realize in other embodiments.Therefore, the present invention can not be restricted to these embodiments shown in this article, but will meet the widest scope consistent with principle disclosed herein and features of novelty.
Claims (5)
1. a CRP latex enhancing immune turbidimetric assay single reagent, it is characterized in that, described CRP latex enhancing immune turbidimetric assay reagent comprises the latex particle of CRP antibody labeling, damping fluid, surfactant, inorganic salts, stabilizing agent, suspending agent, excipient and antiseptic; The regulating power of described damping fluid requires that pH scope is between 7.0-9.0, and the concentration range of described damping fluid is 10-100mmol/L; Described stabilizing agent is BSA, gelatin or casein; Described inorganic salts are a kind of, two or more combination in sodium chloride, potassium chloride, lime chloride, ammonium chloride, sodium sulphate, potassium sulfate; Described surfactant is polysorbas20 or triton x-100; Described excipient is the trehalose of concentration 1%-5%; Described suspending agent is the glycerine of concentration 1%-10%; Described antiseptic is the one in Sodium azide, thimerosal, Proclin-300;
The diameter of described latex particle is 50nm-4000nm; Its finishing group is-COOH ,-NH
2,-SH ,-OH, one in-CHO or physisorption type latex particle.
2. CRP latex enhancing immune turbidimetric assay single reagent according to claim 1, it is characterized in that, the diameter of described latex particle is 100-250nm and finishing group is-COOH or-NH
2in one.
3. CRP latex enhancing immune turbidimetric assay single reagent according to claim 1, it is characterized in that, described damping fluid is 3-[N, N-bis-(hydroxyethyl) is amino]-2-hydroxy-propanesulfonic acid-sodium hydrate buffer solution (DIPSO-NaOH), 4-(2-hydroxyethyl)-1-piperazine propane sulfonic acid-sodium hydrate buffer solution (HEPPS-NaOH), 3-(N-morpholinyl) propane sulfonic acid-sodium hydrate buffer solution (MOPS-NaOH), N-(2-hydroxyethyl) piperazine-N'-2-ethane sulfonic acid-NaOH damping fluid (HEPES-NaOH), trishydroxymethylaminomethane-HCl damping fluid (Tris-HCl), phosphate buffer, imidazole buffer, glycine-NaOH buffer, one in barbitol buffer solution.
4. CRP latex enhancing immune turbidimetric assay single reagent according to claim 1, it is characterized in that, described antiseptic is Sodium azide, and concentration is 0.1%.
5. a single reagent CRP latex enhancing immune turbidimetric assay kit, it is characterized in that, described single reagent CRP latex enhancing immune turbidimetric assay kit comprises at least one detector tube, is loaded with the arbitrary described CRP latex enhancing immune turbidimetric assay single reagent of claim 1 to 4 in detector tube.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101135687A (en) * | 2005-10-23 | 2008-03-05 | 王贤理 | Reagent kit for inspecting apoAI and apoB |
| CN101368947A (en) * | 2008-09-24 | 2009-02-18 | 王贤理 | Immune globulin A detection reagent |
| CN101881777A (en) * | 2010-06-30 | 2010-11-10 | 深圳市国赛生物技术有限公司 | Assay method of high sensitivity C-reactive protein (HS-CRP) and HS-CRP assay kit |
| CN101893625A (en) * | 2009-05-19 | 2010-11-24 | 上海捷门生物技术合作公司 | Kit for testing C-reactive protein Hs-CRP (High-sensitivity C-reactive protein) |
| CN101893619A (en) * | 2010-02-10 | 2010-11-24 | 上海蓝怡科技有限公司 | Method for improving stability of latex suspension liquid |
| CN101907628A (en) * | 2009-06-08 | 2010-12-08 | 武汉中太生物技术有限公司 | Reagent kit for measuring content of C-reactive protein in whole blood and measuring method thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101000349B (en) * | 2006-12-31 | 2011-08-24 | 浙江伊利康生物技术有限公司 | Kit for testing prealbumin |
-
2013
- 2013-03-13 CN CN201310079651.XA patent/CN104049085B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101135687A (en) * | 2005-10-23 | 2008-03-05 | 王贤理 | Reagent kit for inspecting apoAI and apoB |
| CN101368947A (en) * | 2008-09-24 | 2009-02-18 | 王贤理 | Immune globulin A detection reagent |
| CN101893625A (en) * | 2009-05-19 | 2010-11-24 | 上海捷门生物技术合作公司 | Kit for testing C-reactive protein Hs-CRP (High-sensitivity C-reactive protein) |
| CN101907628A (en) * | 2009-06-08 | 2010-12-08 | 武汉中太生物技术有限公司 | Reagent kit for measuring content of C-reactive protein in whole blood and measuring method thereof |
| CN101893619A (en) * | 2010-02-10 | 2010-11-24 | 上海蓝怡科技有限公司 | Method for improving stability of latex suspension liquid |
| CN101881777A (en) * | 2010-06-30 | 2010-11-10 | 深圳市国赛生物技术有限公司 | Assay method of high sensitivity C-reactive protein (HS-CRP) and HS-CRP assay kit |
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