CN110095615A - A kind of kit measuring hs-CRP content - Google Patents
A kind of kit measuring hs-CRP content Download PDFInfo
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- CN110095615A CN110095615A CN201910447121.3A CN201910447121A CN110095615A CN 110095615 A CN110095615 A CN 110095615A CN 201910447121 A CN201910447121 A CN 201910447121A CN 110095615 A CN110095615 A CN 110095615A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
Abstract
The invention discloses a kind of kits for measuring hs-CRP content; the kit includes R1 reagent and R2 reagent; R2 reagent adds Fucoidan; the ingredient has stabilization to the bovine serum albumin bletilla antibody in reagent; to more effectively protect antibody, reagent performance is optimized.This kit measures hs-CRP content using latex enhancing immune scattered light urbidmetry, and measurement result stability is good, sensitivity is good, expands hs-CRP setting-out line range.
Description
Technical field
The present invention relates to a kind of kits for measuring hs-CRP content, belong to bioassay field.
Background technique
C reactive protein (C-reactive protein, CRP) is infected in body or when tissue damage in blood plasma
Some protein (acute protein) steeply risen, activating complement play opsonic action with the phagocytosis for reinforcing phagocyte, remove
Invade pathogenic microorganism and the damage of body, necrosis, the histocyte of apoptosis.
CRP is an extremely sensitive index of acute-phase response.In acute myocardial infarction AMI, cancer infiltration, wound, surgery
CRP concentration increases rapidly in blood plasma when operation, infection, inflammation, up to 2000 times of normal level.In conjunction with clinical medical history, especially
Facilitate the follow-up course of disease in inflammatory process.
There are two kinds (common CRP and super quick CRP) in current country immunoturbidimetry reagent C RP, and common CRP has higher
Linear but sensitivity is bad, and super quick CRP has higher sensitivity but linear lower, and there have been a kind of two kinds of names of reagent in this way
Claim, purposes also different from；Slightly raising is related to coronary artery events, apoplexy and peripheral angiopathy for super quick CRP (Hs-CRP),
It is an independent risk factor；HS-CRP has been found to be the independent hazard factor for causing cardiovascular disease by chronic inflammation,
Its concentration is detected to play an important role to the intervention and prognosis of cardiovascular disease and paid attention to by clinic.
CRP detection kit in the market haves the shortcomings that accuracy is low, the range of linearity is narrow, stability is poor.The present invention
The kit of offer uses latex Immune scatter turbidimetry, and Fucoidan is added in latex antibody R2 reagent as stabilization
Agent, to improve the accuracy and stability of kit, has widened kit so that antibody is more stable in the kit
The range of linearity.
Summary of the invention
The present invention overcomes above-mentioned the deficiencies in the prior art, provide a kind of kit for measuring hs-CRP content.
The present invention adds Fucoidan, measures hs-CRP content, measurement knot using latex enhancing immune scattered light urbidmetry
Fruit stability is good, sensitivity is good, expands hs-CRP setting-out line range.
A kind of kit measuring hs-CRP content, including buffer R1 reagent and latex antibody R2 reagent；
The kelp fucan of 15mg/mL (final concentration of the ingredient in reagent) is added in the latex antibody R2 reagent
Sugar.
Further, above-mentioned buffer R1 reagent includes that (following each component concentration are that the component is being tried to following component
Final concentration in agent): 0.04-0.06mol/L 3- bis- (2- ethoxys) amino -2- hydroxy-propanesulfonic acid (DIPSO) buffer,
0.10-0.15mol/L sodium chloride, 0.1-0.2% triton x-100,0.8-1.0% Macrogol 6000,0.01-0.02%
ProClin300。
Further, above-mentioned latex antibody R2 reagent also comprises the following components that (following each component concentration are that the component exists
Final concentration in reagent): 0.4-0.6mol/L phosphate buffer, 0.4-0.6mol/L MES buffer, 0.25-0.26% are anti-
HCRP polyclonal antibody latex, 0.08-1% bovine serum albumin(BSA), 0.01-0.02%ProClin300.
Further, bis- (2- ethoxy) amino -2- hydroxy-propanesulfonic acid (DIPSO) pH of buffer of above-mentioned 3- are 7.2-7.8,
The phosphate buffer pH is 6.8-7.6, and the MES pH of buffer is 5.6-6.4.
Further, application of the mentioned reagent box in measurement hs-CRP content.
The utility model has the advantages that
The present invention provides a kind of kit for measuring hs-CRP content, which is added to kelp fucan
Sugar, Fucoidan can be used as the antibody and bovine serum albumin(BSA) that stabilizer comes in stable reagent.Fucoidan is one
Kind is rich in the acid polyanionic polysaccharide of sulfate radical, and main chain is fucose, and sulfate is predominantly located at the C2 and/or C4 of fucose
Position and C3 and/or C4 of side chain galactolipin.As it can be seen that Fucoidan main chain and side chain contain with negative electrical charge
Sulfate group.Therefore, Fucoidan stablizes anti crp antibody in R2 reagent by electrostatic repulsion and ox blood is pure
Albumen.
Antibody is easily degraded in low concentration and is inactivated, so bovine serum albumin(BSA) etc usually can all be added in reagent
Unrelated protein as stabilizer, kit provided by the invention adds Fucoidan ingredient, the ingredient in R2 reagent
There is stabilization to the bovine serum albumin bletilla antibody in reagent, to more effectively protect antibody, optimizes agents
Energy (accuracy, linear, stability etc.).
Stabilization of kit provided by the invention is higher than existing similar kit, the range of linearity of detection, accuracy and spirit
Sensitivity is above available reagent box, significantly improves the stability of hs-CRP detection reagent, accuracy and sensitivity,
Hs-CRP setting-out line range is expanded, solves existing hs-CRP detection kit detection sensitivity
Low with accuracy, the range of linearity is narrow, the poor problem of stability.
Detailed description of the invention
Fig. 1 is formulated the linear regression curves that matched kit detects c reactive protein not add Fucoidan.
Fig. 2 is formulated the linear regression curves that matched kit detects c reactive protein for addition Fucoidan.
Fig. 3 is the linear regression curves that kit detects c reactive protein in embodiment 5.
Specific embodiment
Below with reference to embodiment and attached drawing, the present invention will be further described, but the present invention is not by the limit of following embodiments
System.
The preparation of 1 kit of embodiment.
1.1 do not add the preparation of Fucoidan kit
1.1.1 R1 preparation of reagents
Bis- (2- ethoxy) amino -2- hydroxy-propanesulfonic acid (DIPSO) buffer concentrations of 3- are 0.05mol/L, use 0.1mol/
L HCl solution adjusts pH to 7.5.Add 0.15mol/L sodium chloride, 0.1% triton x-100,1.0% Macrogol 6000
And 0.02%ProClin300.Wherein, DIPSO buffer plays the role of stable pH value of reaction system, and sodium chloride is reaction
System provides ionic environment, and triton x-100 and Macrogol 6000 are surfactant, and ProClin 300 is used as preservative
It works in the reaction system.
1.1.2 R2 preparation of reagents
By taking 1L produces volume as an example:
(1) preparation of 0.5mol/L phosphate buffer (pH7.2)
1. purified water 2L needed for measuring；
2. weighing NaH2PO42H2O 68.6444g (0.22mol/L) and Na2HPO412H2O 200.5584g respectively
(0.28mol/L) is added in 1L purified water, at room temperature sufficiently dissolution, adjusts pH to 7.2 with sodium hydroxide.
(2) preparation of 0.5mol/L MES buffer (pH6.0)
1. purified water 1L needed for measuring；
2. weighing 2-morpholine ethane sulfonic acid 97.6g, it is added in 1L purified water, sufficiently dissolution, is adjusted with sodium hydroxide at room temperature
PH to 6.0；(0.5mol/L).
3. the amount of preparation of MES buffer is the polystyrene latex pearl of 10 times of volumes in production process.
(3) weighing of unclassified stores
1. Sulfo-NHS: weighing Sulfo-NHS 2g, the purified water stirring and dissolving of 2 times of bulking values is added, stir
5min dissolves it sufficiently；(0.2%Sulfo-NHS)
2. EDC: weighing EDC 1.4g, the purified water stirring and dissolving of 2 times of bulking values is added；Stirring 5min keeps it sufficiently molten
Solution；(0.14%EDC)
3. polystyrene latex pearl: measuring polystyrene latex pearl 2.5mL；(0.25% polystyrene latex pearl)
4. ethanol amine: measuring ethanol amine 1mL；(0.1% ethanol amine)
5. anti-human c reactive protein polyclonal antibody: measuring anti-human 83.3 μ L of c reactive protein polyclonal antibody；(1mL polyphenyl
The anti-human c reactive protein polyclonal antibody of 1mg is added in ethylene latex bead (measuring volume))
6. bovine serum albumin(BSA): weighing bovine serum albumin(BSA) 10g；(1% bovine serum albumin(BSA))
7. ProClin300: measuring ProClin300 0.2mL.(0.02%ProClin 300)
(4) activation of polystyrene latex microballoon
It is uniformly mixed 1. the EDC solution of dissolution is added in Sulfo-NHS solution；
2. the polystyrene latex pearl of measurement is added in the mixed solution of the EDC and Sulfo-NHS that have dissolved, add
It is (poly- that 5 times of volumes are then added in 2.5mL 0.5mol/L MES buffer (pH6.0) (with polystyrene latex pearl same volume)
Polystyrene latex pearl) purified water sufficiently dissolved, and it reacts at room temperature 30 minutes, is stirred continuously.
3. prepared polystyrene latex solution is transferred in centrifugal bottle, it is centrifuged after trim, 15000rpm, 8 degree, 50
Minute；
4. abandoning supernatant, centrifugation is collected；40mL phosphate buffer is added, polystyrene cream is sufficiently resuspended with egg-whisk
Glue bead is centrifuged, 15000rpm after trim, and 8 degree, 40 minutes；Twice in succession；
5. abandoning supernatant, centrifugation is collected；40mL phosphate buffer suspension polystyrene latex pearl again, ultrasound is added
(150W 3 seconds, is spaced 10 seconds, 90 times) makes its dispersion within 20 minutes.
(5) coupling and closing of antibody
1. the anti-human c reactive protein polyclonal antibody of measurement is quickly added at one time in polystyrene colloidal milk solution；Add
Enter 40mL phosphate buffer, stir evenly, is placed at room temperature for 30min；Then beaker is moved to 2-8 DEG C overnight.
2. second day, in the antibody latex solution after coupling, ethanol amine being added, stirs 30min；
3. 15000rmp is centrifuged 50 minutes, supernatant is abandoned.It is washed twice with phosphate buffer, after centrifugation, abandons supernatant；
It is suspended again anti-human c reactive protein polyclonal antibody latex with 40mL phosphate buffer, ultrasound 20 minutes (150W, 3 seconds,
Every 10 seconds, 90 times)；
4. bovine serum albumin(BSA) and ProClin300 are added into antibody latex, after completely dissolution, 960mL phosphate is added
Buffer is to get R2 reagent.
(1) purified water 2L is measured, weighs potassium dihydrogen phosphate 27.218g and sodium chloride 58.44g respectively, 2L purified water is added
In, sufficiently dissolution, obtains phosphate buffer at room temperature.
(2) 100mL phosphate buffer is added in the polystyrene latex particulate that 40mL partial size is 300nm, then plus
Enter the polystyrene latex particulate that 10mL partial size is 60nm；
(3) NHS 0.02g is added sufficiently to dissolve, adds 0.05g EDC, react at room temperature 30 minutes, be stirred continuously；
(4) 12000rpm is centrifuged 30 minutes, abandons supernatant, collects centrifugation；It is washed twice, is centrifuged with phosphate buffer
After precipitating, supernatant is abandoned；
(5) it is suspended again polystyrene latex particulate with 100mL phosphate buffer, ultrasound makes its dispersion in 30 minutes；
(6) the anti-human d-dimer antibody of 0.581mL is dissolved in phosphate buffer, above-mentioned activated polyphenyl second is added
Alkene latex particle solution is coated with 2h；
(7) in the antibody latex being coated with, 5mL ethanol amine is added, stirs 10 minutes；
(8) 12000rpm is centrifuged 30 minutes, abandons supernatant.It is washed twice with phosphate buffer, after centrifugation, in abandoning
Clearly.It is suspended anti-human d-dimer antibody latex, is dispersed again with 100mL phosphate buffer within ultrasound 20 minutes；
(9) 10g BSA and 0.2g ProClin 300 is added and the phosphate buffer of 900mL is added after completely dissolution,
Up to R2 reagent.
The preparation of 1.2 addition Fucoidan kits
1.2.1 R1 preparation of reagents
Bis- (2- ethoxy) amino -2- hydroxy-propanesulfonic acid (DIPSO) buffer concentrations of 3- are 0.05mol/L, use 0.1mol/
L HCl solution adjusts pH to 7.5.Add 0.15mol/L sodium chloride, 0.1% triton x-100,1.0% Macrogol 6000
And 0.02%ProClin300.Wherein, DIPSO buffer plays the role of stable pH value of reaction system, and sodium chloride is reaction
System provides ionic environment, and triton x-100 and Macrogol 6000 are surfactant, and ProClin 300 is used as preservative
It works in the reaction system.
1.2.2 R2 preparation of reagents
By taking 1L produces volume as an example:
(1) preparation of 0.5mol/L phosphate buffer (pH7.2)
1. purified water 2L needed for measuring；
2. weighing NaH2PO42H2O 68.6444g (0.22mol/L) and Na2HPO412H2O 200.5584g respectively
(0.28mol/L) is added in 1L purified water, at room temperature sufficiently dissolution, adjusts pH to 7.2 with sodium hydroxide.
(2) preparation of 0.5mol/L MES buffer (pH6.0)
1. purified water 1L needed for measuring；
2. weighing 2-morpholine ethane sulfonic acid 97.6g, it is added in 1L purified water, sufficiently dissolution, is adjusted with sodium hydroxide at room temperature
PH to 6.0；(0.5mol/L).
3. the amount of preparation of MES buffer is the polystyrene latex pearl of 10 times of volumes in production process.
(3) weighing of unclassified stores
1. Sulfo-NHS: weighing Sulfo-NHS 2g, the purified water stirring and dissolving of 2 times of bulking values is added, stir
5min dissolves it sufficiently；(0.2%Sulfo-NHS)
2. EDC: weighing EDC 1.4g, the purified water stirring and dissolving of 2 times of bulking values is added；Stirring 5min keeps it sufficiently molten
Solution；(0.14%EDC)
3. polystyrene latex pearl: measuring polystyrene latex pearl 2.5mL；(0.25% polystyrene latex pearl)
4. ethanol amine: measuring ethanol amine 1mL；(0.1% ethanol amine)
5. anti-human c reactive protein polyclonal antibody: measuring anti-human 83.3 μ L of c reactive protein polyclonal antibody；(1mL polyphenyl
The anti-human c reactive protein polyclonal antibody of 1mg is added in ethylene latex bead (measuring volume))
6. bovine serum albumin(BSA): weighing bovine serum albumin(BSA) 10g；(1% bovine serum albumin(BSA))
7. ProClin300: measuring ProClin300 0.2mL.(0.02%ProClin 300)
(4) activation of polystyrene latex microballoon
It is uniformly mixed 1. the EDC solution of dissolution is added in Sulfo-NHS solution；
2. the polystyrene latex pearl of measurement is added in the mixed solution of the EDC and Sulfo-NHS that have dissolved, add
It is (poly- that 5 times of volumes are then added in 2.5mL 0.5mol/L MES buffer (pH6.0) (with polystyrene latex pearl same volume)
Polystyrene latex pearl) purified water sufficiently dissolved, and it reacts at room temperature 30 minutes, is stirred continuously.
3. prepared polystyrene latex solution is transferred in centrifugal bottle, it is centrifuged after trim, 15000rpm, 8 degree, 50
Minute；
4. abandoning supernatant, centrifugation is collected；40mL phosphate buffer is added, polystyrene cream is sufficiently resuspended with egg-whisk
Glue bead is centrifuged, 15000rpm after trim, and 8 degree, 40 minutes；Twice in succession；
5. abandoning supernatant, centrifugation is collected；40mL phosphate buffer suspension polystyrene latex pearl again, ultrasound is added
(150W 3 seconds, is spaced 10 seconds, 90 times) makes its dispersion within 20 minutes.
(5) coupling and closing of antibody
1. the anti-human c reactive protein polyclonal antibody of measurement is quickly added at one time in polystyrene colloidal milk solution；Add
Enter 40mL phosphate buffer, stir evenly, is placed at room temperature for 30min；Then beaker is moved to 2-8 DEG C overnight.
2. second day, in the antibody latex solution after coupling, ethanol amine being added, stirs 30min；
3. 15000rmp is centrifuged 50 minutes, supernatant is abandoned.It is washed twice with phosphate buffer, after centrifugation, abandons supernatant；
It is suspended again anti-human c reactive protein polyclonal antibody latex with 40mL phosphate buffer, ultrasound 20 minutes (150W, 3 seconds,
Every 10 seconds, 90 times)；
4. bovine serum albumin(BSA) and ProClin300 are added into antibody latex, after completely dissolution, 960mL phosphate is added
Buffer is to get R2 reagent.
(1) purified water 2L is measured, weighs potassium dihydrogen phosphate 27.218g and sodium chloride 58.44g respectively, 2L purified water is added
In, sufficiently dissolution, obtains phosphate buffer at room temperature.
(2) 100mL phosphate buffer is added in the polystyrene latex particulate that 40mL partial size is 300nm, then plus
Enter the polystyrene latex particulate that 10mL partial size is 60nm；
(3) NHS 0.02g is added sufficiently to dissolve, adds 0.05g EDC, react at room temperature 30 minutes, be stirred continuously；
(4) 12000rpm is centrifuged 30 minutes, abandons supernatant, collects centrifugation；It is washed twice, is centrifuged with phosphate buffer
After precipitating, supernatant is abandoned；
(5) it is suspended again polystyrene latex particulate with 100mL phosphate buffer, ultrasound makes its dispersion in 30 minutes；
(6) the anti-human d-dimer antibody of 0.581mL is dissolved in phosphate buffer, above-mentioned activated polyphenyl second is added
Alkene latex particle solution is coated with 2h；
(7) in the antibody latex being coated with, 5mL ethanol amine is added, stirs 10 minutes；
(8) 12000rpm is centrifuged 30 minutes, abandons supernatant.It is washed twice with phosphate buffer, after centrifugation, in abandoning
Clearly.It is suspended anti-human d-dimer antibody latex, is dispersed again with 100mL phosphate buffer within ultrasound 20 minutes；
(9) 10g BSA, 15g Fucoidan and 0.2g ProClin 300 is added, after completely dissolution, 900mL is added
Phosphate buffer to get R2 reagent.
Embodiment 2 using the kit measurement hs-CRP content obtained in embodiment 1 method
1) preparation of CRP calibration object: with phosphate buffer (pH7.5,0.1mol/L), calf serum (20%), C reaction
Proteantigen is that main raw material(s) prepares CRP calibration object, which is 300.00mg/L；
2) using the Shandong Ai Keda Chinese mugwort Kodak full-automatic specific protein analyzer, instrument parameter: detection temperature: 37 DEG C；Inspection
Survey wavelength: 650nm；Reaction time: 10min；
3) using the CRP calibration object calibration instrument obtained in step 1), calibration curve is made；
4) 200 μ L of R1 reagent, the 100 μ L of R2 reagent for taking kit respectively, take 5 μ L of sample to be tested；
5) it sets instrument parameter and draws standard curve, it is specified that the reagent taken in step 4) and sample are put into instrument
It is tested behind position.
Embodiment 3 is formulated matched kit measurement c reactive protein using Fucoidan is not added.
It is measured using Shandong Chinese mugwort Kodak full-automatic specific protein analyzer, testing conditions are as follows: detection temperature: 37
℃；Detection wavelength: 650nm；Reaction time: 10min；Reagent dosage: R1 reagent: 200 μ L, R2 reagents: 100 μ L；Sample dosage:
5μL。
3.1 accuracy
Not adding the test of kit made from Fucoidan formula has card reference material (ERM-DA474/IFCC), point
It is not positioned proximate to two concentration of 5mg/L and 20mg/L, each sample replication 3 times, test result is denoted as (Xi), by formula
(1) it calculates separately relative deviation (Bi), the relative deviation of 3 results should be no more than ± 15%
Bi=(Xi-T)/T × 100% ... ... ... ... (1)
In formula:
Bi -- relative deviation；
Xi -- measurement concentration；
T -- calibration concentration.
The accuracy of kit in 1 embodiment 1 of table
The relative deviation of acquired results sample 1 is 2.01%, and the relative deviation of sample 2 is 1.18%, shows not add sea
It is preferable that band fucosan is formulated kit accuracy obtained.
3.2 linear
It is diluted to 6 concentration by a certain percentage with the high level sample close to the linearly interval upper limit, such as table 2, wherein low value is dense
The sample of degree must be close to the lower limit of linearly interval.To sample replication 3 times of each concentration, its average value is calculated, will be measured
The average value and dilution ratio of concentration carry out straight line fitting with least square method, obtain equation of linear regression, and calculate linear phase
Relationship number r and absolute deviation or relative deviation；In 0.5-240.0mg/L linearly interval, linearly dependent coefficient | r | it should be not less than
0.990。
2 diluted concentration table of table
| Dilution (mL) | 0 | 0.67 | 0.89 | 0.963 | 0.9877 | 0.9984 |
| Enriched sample (mL) | 1 | 0.33 | 0.11 | 0.037 | 0.0123 | 0.0016 |
Table 3 does not add the range of linearity that Fucoidan is formulated kit obtained
Acquired results equation of linear regression is y=241.16x+0.3088, and linear regression curves are as shown in Figure 1, phase relation
Number r is 0.9998, shows that not adding Fucoidan is formulated kit obtained good relationship in the linear range.
3.3 stability
It is 9.5mg/L with the test of kit made from Fucoidan formula target value is not added, target value range is 7.60-
The quality-control product of 11.40mg/L every retest in 5 days 3 times, calculates average value, until averaging of income value is not within the scope of value.
Table 3 does not add the stability that Fucoidan is formulated kit obtained
| Stability | 1 | 2 | 3 | x |
| 1st day | 9.54 | 9.52 | 9.48 | 9.51 |
| 6th day | 9.46 | 9.52 | 9.48 | 9.49 |
| 11st day | 9.47 | 9.51 | 9.51 | 9.50 |
| 16th day | 9.46 | 9.49 | 9.47 | 9.47 |
| 21st day | 9.53 | 9.52 | 9.49 | 9.51 |
| 26th day | 9.54 | 9.52 | 9.51 | 9.52 |
| 31st day | 9.46 | 9.45 | 9.48 | 9.46 |
| 36th day | 9.48 | 9.47 | 9.51 | 9.49 |
| 41st day | 7.28 | 7.34 | 7.42 | 7.35 |
Preceding 36 days acquired results are within the scope of value, and acquired results show not not within the scope of value at the 41st day
Kit made from addition Fucoidan formula can be stablized 36 days, and the stabilization of kit is preferable.
Embodiment 4 is formulated matched kit measurement c reactive protein using addition Fucoidan.
It is measured using Shandong Chinese mugwort Kodak full-automatic specific protein analyzer, testing conditions are as follows: detection temperature: 37
℃；Detection wavelength: 650nm；Reaction time: 10min；Reagent dosage: R1 reagent: 200 μ L, R2 reagents: 100 μ L；Sample dosage:
5μL。
4.1 accuracy
Kit test made from addition Fucoidan formula has card reference material (ERM-DA474/IFCC), respectively
Two concentration of 5mg/L and 20mg/L are positioned proximate to, each sample replication 3 times, test result is denoted as (Xi), by formula (1)
It calculates separately relative deviation (Bi), the relative deviation of 3 results should be no more than ± 15%
Bi=(Xi-T)/T × 100% ... ... ... ... (1)
In formula:
Bi -- relative deviation；
Xi -- measurement concentration；
T -- calibration concentration.
Table 4 adds the accuracy that Fucoidan is formulated kit obtained
The relative deviation of acquired results sample 1 is 0.19%, and the relative deviation of sample 2 is 0.08%, shows to add kelp
The kit accuracy obtained of fucosan formula is preferable, and is better than and does not add Fucoidan formula kit obtained
Accuracy.4.2 linear
It is diluted to 6 concentration by a certain percentage with the high level sample close to the linearly interval upper limit, such as following table, wherein low value is dense
The sample of degree must be close to the lower limit of linearly interval.To sample replication 3 times of each concentration, its average value is calculated, will be measured
The average value and dilution ratio of concentration carry out straight line fitting with least square method, obtain equation of linear regression, and calculate linear phase
Relationship number r and absolute deviation or relative deviation；In 0.5-240.0mg/L linearly interval, linearly dependent coefficient | r | it should be not less than
0.990。
5 diluted concentration of table
| Dilution (mL) | 0 | 0.67 | 0.89 | 0.963 | 0.9877 | 0.9984 |
| Enriched sample (mL) | 1 | 0.33 | 0.11 | 0.037 | 0.0123 | 0.0016 |
Table 6 adds the range of linearity that Fucoidan is formulated kit obtained
Acquired results equation of linear regression is y=306.44x+0.2676, and linear regression curves are as shown in Fig. 2, phase relation
Number r is 0.9998, shows that adding Fucoidan is formulated kit obtained good relationship in the linear range, and linear
Range, which is wider than, does not add the range of linearity that Fucoidan is formulated kit obtained.
4.3 stability
The kit test target value made from addition Fucoidan formula is 9.5mg/L, and target value range is 7.60-
The quality-control product of 11.40mg/L every retest in 5 days 3 times, calculates average value, until averaging of income value is not within the scope of value.
Table 7 adds the stability that Fucoidan is formulated kit obtained
| Stability | 1 | 2 | 3 | x |
| 1st day | 9.48 | 9.51 | 9.51 | 9.50 |
| 6th day | 9.51 | 9.49 | 9.49 | 9.50 |
| 11st day | 9.48 | 9.50 | 9.48 | 9.49 |
| 16th day | 9.51 | 9.48 | 9.50 | 9.50 |
| 21st day | 9.47 | 9.46 | 9.50 | 9.48 |
| 26th day | 9.49 | 9.47 | 9.52 | 9.49 |
| 31st day | 9.49 | 9.52 | 9.51 | 9.51 |
| 36th day | 9.53 | 9.52 | 9.92 | 9.66 |
| 41st day | 9.48 | 9.48 | 9.49 | 9.48 |
| 46th day | 7.46 | 7.43 | 7.43 | 7.44 |
Preceding 41 days acquired results are within the scope of value, and acquired results show to add not within the scope of value at the 46th day
Add kit made from Fucoidan formula that can stablize 41 days, the stabilization of kit is preferable, and is better than and does not add kelp
Fucosan is formulated the stability of kit obtained.
The existing latex Immune scatter turbidimetry hs-CRP assay kit on the market of embodiment 5.
It is measured using commercially available latex Immune scatter turbidimetry hs-CRP assay kit, it is accuracy, linear
Measuring method with stability is as described in embodiment 2,3.
5.1 accuracy
The accuracy of 8 available reagent box of table
The relative deviation of acquired results sample 1 is 2.78%, and the relative deviation of sample 2 is 2.73%, is greater than embodiment 2,3
Used in kit, the kit accuracy is lower.
5.2 linear
9 available reagent box of table it is linear
The available reagent box range of linearity is 0.5-200mg/L and linear regression curves as shown in figure 3, its related coefficient is
0.9996, narrower than the kit range of linearity used in embodiment 2,3, correlation is poor.
5.3 stability
The stability of 10 available reagent box of table
31 days acquired results are within the scope of value before available reagent box, and acquired results are not value range at the 36th day
It is interior, show that available reagent box can be stablized 31 days, stability is lower than kit used in embodiment 2,3.
Kit accuracy made from Fucoidan formula is not added is significantly higher than available reagent box；Range of linearity ratio
Available reagent box is wide, and correlation is good；Stability is apparently higher than available reagent box；It adds Fucoidan and is formulated reagent obtained
Box accuracy, which is significantly higher than, does not add Fucoidan formula kit obtained；Range of linearity ratio does not add kelp fucan
Sugar formula kit obtained is wide, and correlation is good；Stability, which is apparently higher than, does not add Fucoidan formula reagent obtained
Box；Show to add the range of linearity that kit made from Fucoidan formula has widened hs-CRP detection, significantly
Improve its accuracy and stability.
The present invention provides a kind of kits of hs-CRP content for adding Fucoidan, including buffering
Liquid R1 reagent and latex antibody R2 reagent are added to Fucoidan as stabilizer in the R2 reagent.The present invention provides
Kit use latex Immune scatter turbidimetry, accuracy be higher than similar kit, the range of linearity is wider, and correlation is good；It is aobvious
The stability for improving hs-CRP detection is write, it is low to solve existing hs-CRP detection kit accuracy,
The problem of stability difference.
Claims (6)
1. a kind of kit for measuring hs-CRP content, which is characterized in that including buffer R1 reagent and latex antibody
R2 reagent；
The Fucoidan of final concentration of 15mg/mL is added in the latex antibody R2 reagent.
2. kit as described in claim 1, which is characterized in that the buffer R1 reagent includes following component, following
Each component concentration is final concentration of the component in R1 reagent: bis- (2- ethoxy) amino -2- hydroxyls of 0.04-0.06mol/L3-
Base propane sulfonic acid buffer, 0.10-0.15mol/L sodium chloride, 0.1-0.2% triton x-100,0.8-1.0% polyethylene glycol
6000,0.01-0.02%ProClin300.
3. kit as described in claim 1, which is characterized in that the latex antibody R2 reagent also comprises the following components, under
Stating each component concentration is the final concentration in component R2 reagent: 0.4-0.6mol/L phosphate buffer, 0.4-0.6mol/L
MES buffer, the anti-human c reactive protein polyclonal antibody latex of 0.25-0.26%, 0.08-1% bovine serum albumin(BSA), 0.01-
0.02%ProClin300.
4. kit as claimed in claim 2, which is characterized in that bis- (2- ethoxy) amino -2- hydroxy-propanesulfonic acids of 3-
The pH of buffer is 7.2-7.8.
5. kit as claimed in claim 3, which is characterized in that the phosphate buffer pH is 6.8-7.6, the MES
PH of buffer is 5.6-6.4.
6. application of the kit as described in any one in claim 1-5 in measurement hs-CRP content.
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| CN201910447121.3A CN110095615A (en) | 2019-05-27 | 2019-05-27 | A kind of kit measuring hs-CRP content |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910447121.3A CN110095615A (en) | 2019-05-27 | 2019-05-27 | A kind of kit measuring hs-CRP content |
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| CN110095615A true CN110095615A (en) | 2019-08-06 |
Family
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| CN201910447121.3A Pending CN110095615A (en) | 2019-05-27 | 2019-05-27 | A kind of kit measuring hs-CRP content |
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006184131A (en) * | 2004-12-27 | 2006-07-13 | Nippon Meat Packers Inc | Measuring method of fucoidan content and fucoidan-containing food |
| CN101285826A (en) * | 2008-05-23 | 2008-10-15 | 中国海洋大学 | Sea pumpkin and sea pumpkin product sea pumpkin polysaccharide content determination method |
| CN101377504A (en) * | 2008-04-16 | 2009-03-04 | 北京科美东雅生物技术有限公司 | Chemiluminescence immune analysis determination reagent kit for detecting Toxoplasma Gondi IgM antibody |
| CN101769932A (en) * | 2010-01-26 | 2010-07-07 | 宁波美康生物科技有限公司 | Full-range C-reactive protein detection kit |
| CN105158476A (en) * | 2015-06-03 | 2015-12-16 | 南京闻智生物科技有限公司 | Full-scale range C-reactive protein latex-enhanced immunoturbidimetry detection kit |
| CN108303544A (en) * | 2017-01-13 | 2018-07-20 | 深圳开立生物医疗科技股份有限公司 | A kind of whole blood c reactive protein detection kit |
| CN109073641A (en) * | 2016-04-13 | 2018-12-21 | 美迪恩斯生命科技株式会社 | Use the Radioimmunoassay of vascular endothelial growth of sulfated polysaccharides class |
| CN109310628A (en) * | 2016-06-30 | 2019-02-05 | 赛特瑞恩股份有限公司 | Stable liquid pharmaceutical formulation |
-
2019
- 2019-05-27 CN CN201910447121.3A patent/CN110095615A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006184131A (en) * | 2004-12-27 | 2006-07-13 | Nippon Meat Packers Inc | Measuring method of fucoidan content and fucoidan-containing food |
| CN101377504A (en) * | 2008-04-16 | 2009-03-04 | 北京科美东雅生物技术有限公司 | Chemiluminescence immune analysis determination reagent kit for detecting Toxoplasma Gondi IgM antibody |
| CN101285826A (en) * | 2008-05-23 | 2008-10-15 | 中国海洋大学 | Sea pumpkin and sea pumpkin product sea pumpkin polysaccharide content determination method |
| CN101769932A (en) * | 2010-01-26 | 2010-07-07 | 宁波美康生物科技有限公司 | Full-range C-reactive protein detection kit |
| CN105158476A (en) * | 2015-06-03 | 2015-12-16 | 南京闻智生物科技有限公司 | Full-scale range C-reactive protein latex-enhanced immunoturbidimetry detection kit |
| CN109073641A (en) * | 2016-04-13 | 2018-12-21 | 美迪恩斯生命科技株式会社 | Use the Radioimmunoassay of vascular endothelial growth of sulfated polysaccharides class |
| CN109310628A (en) * | 2016-06-30 | 2019-02-05 | 赛特瑞恩股份有限公司 | Stable liquid pharmaceutical formulation |
| CN108303544A (en) * | 2017-01-13 | 2018-07-20 | 深圳开立生物医疗科技股份有限公司 | A kind of whole blood c reactive protein detection kit |
Non-Patent Citations (1)
| Title |
|---|
| 杜慧等: "海带岩藻聚糖的蛋白质稳定及抗油脂酸败作用", 《食品安全质量检测学报》 * |
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