CN106501536A - A kind of latex orientation coupling technology detection lipoprotein（a）Test kit - Google Patents
A kind of latex orientation coupling technology detection lipoprotein（a）Test kit Download PDFInfo
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Abstract
The present invention provides a kind of test kit of latex orientation coupling technology detection lipoprotein (a), the test kit principle is based on latex enhancing immune turbidimetry, its feature is to orient coupling technology using antibody, the aminopolystyrene latex microsphere that two kinds of particle diameters are activated respectively by N N-Hydroxysuccinimide/maleimide heterobifunctional crosslinker, reducing process reduces anti-human lipoprotein (a) monoclonal antibody, the latex microsphere of two kinds of particle diameters after activation is made to be coupled with the anti-Lp(a) monoclonal antibody orientation after reduction respectively, form anti-human lipoprotein (a) latex particle, antibody is made to connect microsphere with Fc areas, Fab areas flex outward, realize efficiently being combined with sample antigens, in 37 DEG C of constant temperature, wavelength is under 600nm, mensuration absorbance, calculate sample lipoprotein (a) content.Compared with traditional method, this test kit antibody utilization rate is high, and sensitivity is high, good linearity, energy effective control difference between batch, is worth further genralrlization to use.
Description
Technical field：
The present invention relates to medical science detection and in-vitro diagnosis field, and in particular to a kind of latex orientation coupling technology detection fat egg
The test kit of (a) in vain.
Background technology：
Lipoprotein (a) is a kind of special independent plasma lipoprotein, is similar to the lipid of low density lipoprotein, LDL (LDL), is
Norway geneticist Berg in 1963 is had found when the Genetic Variations of low density lipoprotein, LDL are studied, mainly after liver synthesis
Blood is secreted into, the change of its content has very important clinical meaning in the diseases such as thrombotic disease, kidney disease, diabetes
Justice.
The conventional method of lipoprotein (a) detection technique has several as follows in the market：Single immunodiffusion (SRID), put
Penetrate immunoassay (RIA), fluorescence immunoassay and send out (FIA), time-resolved fluorescent immunoassay (TRFIA), enzyme linked immunological
Algoscopy (ELISA), latex particle method, granule strengthen transmission, immunoturbidimetry (PETIA), granule enhancing scattering immunoturbidimetry
Method (PENIA), Latex-enhanced immunoturbidimetric assay etc., in the majority with latex enhancing immune turbidimetry.Latex enhancing immune is than turbid
Deriving technology in common immunoturbidimetry technology owned by France, overcomes the very limited amount of shortcoming of common immunoturbidimetry technology semaphore,
Make antibody volume have the growth on the order of magnitude, greatly improve detectability, become a kind of easy to be quick, the high inspection of sensitivity
Survey method, but the method to there is also the direction that huge technological difficulties, i.e. antibody is connected on latex microsphere uncontrollable so that
Its Activity and stabill (including do not change with the change of time and batch between concordance) it cannot be guaranteed that.Therefore, one is found
Plant the method that coupling is oriented between antibody and latex microsphere, directions that both are connected are controllable, are the technology of this area urgent need to resolve
Problem.
Content of the invention：
In view of above technical problem, it is an object of the invention to propose a kind of latex orientation coupling technology detection lipoprotein
A the test kit of (), based on latex enhancing immune turbidimetry, orients coupling technology with antibody, by N- hydroxysuccinimidyl acyls
Imines/maleimide heterobifunctional crosslinker activates the aminopolystyrene latex microsphere of two kinds of particle diameters respectively, and reducing process is also
Former anti-human lipoprotein (a) monoclonal antibody, make the latex microsphere of two kinds of particle diameters after activation respectively with reduction after anti-human fat egg
(a) monoclonal antibody orientation is coupled in vain, forms anti-human lipoprotein (a) latex particle, makes antibody connect microsphere, Fab areas with Fc areas
Flex outward, realize efficiently being combined with sample antigens.
The technical scheme is that：Using N-hydroxy-succinamide/maleimide heterobifunctional crosslinker as two
The activator of the aminopolystyrene latex microsphere of particle diameter is planted, activated state latex microsphere is formed；Reducing process reduces anti-human lipoprotein
A () monoclonal antibody, is made antibody disulfide bond be unfolded into free sulfhydryl groups, is coupled with activated state latex microsphere, formed stable
Thioether bond, so as to realize orientation be coupled.Detection kit is prepared based in this approach, and reagent 1 includes buffer, stabilizer
And preservative, reagent 2 includes anti-human lipoprotein (a) latex microsphere, buffer, protective agent, stabilizer and preservative.
Reagent 1 described in test kit of the present invention includes but is not limited to Tris-HCL buffer, PBS with the buffer in reagent 2
Buffer, disodium hydrogen phosphate-citrate buffer solution, one or more in potassium dihydrogen phosphate-sodium hydrate buffer solution, more preferably
PBS.PH scopes are 6.5-8.5, more preferably PH6.5-7.5.
Stabilizer in reagent 1 described in test kit of the present invention includes but is not limited to Macrogol 8000, sucrose, glycerol, Portugal
Grape sugar, gelatin, sodium chloride, potassium chloride, sodium carbonate, sodium sulfate, more preferably one or more in potassium sulfate, Polyethylene Glycol
8000.
Preservative in reagent 1 described in test kit of the present invention and reagent 2 includes but is not limited to sodium azide, proclin 300,
One or more in thimerosal.
Protective agent in reagent 2 described in test kit of the present invention is EDTA.
Stabilizer in reagent 2 described in test kit of the present invention includes but is not limited to Macrogol 8000, sucrose, glycerol, Portugal
Grape sugar, gelatin, sodium chloride, potassium chloride, sodium carbonate, sodium sulfate, more preferably one or more in potassium sulfate, sucrose.
Further, with above-mentioned preferred material reagent preparation box, (liquid double reagent, R1: R2 is 4: 1), concentration is respectively
Anti-human lipoprotein (a) latex microsphere in reagent 2 described in test kit of the present invention is prepared as follows：
1) activation of aminopolystyrene latex microsphere
Aminopolystyrene latex microsphere and N- hydroxysuccinimidyl that concentration be the two kind particle diameters of 2.5% (w/v) are taken respectively
Acid imide/maleimide heterobifunctional crosslinker, is mixed in PBS (20mmol/ by rate of charge for 1: 5 (weight ratio)
L, PH7.0) in, 25 DEG C of activation 30-60min, high speed centrifugation (10000rpm, 30min) remove supernatant, use PBS
(20mmol/L, PH7.0) washes twice the unnecessary cross-linking agent of removal, is made into PBS (20mmol/L, PH7.0) after washing
1% (w/v) mixed liquor, 25 DEG C of concussion 10-20min are resuspended, obtain activation latex microsphere.
Described aminopolystyrene latex microsphere particle diameter selects 100-200nm respectively, two kinds of specifications of 200-300nm, more
Preferable particle size is respectively 110nm and 200m.Particle fraction is 20: 80-80: 20, more preferably 60: 40.
Described N-hydroxy-succinamide/maleimide heterobifunctional crosslinker includes but is not limited to 4- (N- Malaysias
Acid imide methyl) hexamethylene -1- carboxylic acids sulfonic group butanimide ester sodium salt (Sulfo-SMCC), 4- (N- maleimide first
Base) hexamethylene -1- carboxylic acids sulfonic group succinimide ester (SMCC), thiosuccimide base 4- (p- maleimide phenyls)
Butyl ester (Sulfo-SMPB), succinimido 4- (p- maleimide phenyls) butyrate (SMPB), succinimido -6-
[(β-maleimide propionamido-)] own ester (SMPH), m- maleimidobenzoyl-N- hydroxy thiosuccinimide esters
(Sulfo-MBS), N- [κ-maleimide undecanoyl oxygen]-thiosuccimide ester (Sulfo-KMUS), N- [γ-Malaysia
Acid imide butyryl oxygen] succinimide ester (Sulfo-GMBS), N- [ε-maleimide acetyl group oxygen] thiosuccimide
One or more in ester (Sulfo-EMCS), more preferably 4- (N- maleimidomehyls) hexamethylene -1- carboxylic acid sulfonic group ambers
Amber imide ester sodium salt (Sulfo-SMCC).
2) anti-human lipoprotein (a) monoclonal antibody reduction
Anti-human lipoprotein (a) monoclonal antibody and reducing agent (1-10mM) is taken, is mixed for 1: 800 (weight ratio) by rate of charge
In PBS (20mmol/L, PH7.0), 25 DEG C of reduction 30min, it is unnecessary to be removed by 200 chromatography of superdex
Reducing agent, adds iodoacetamide 0.5mg, 37 DEG C of closing sulfydryl 30-45min.
Described for anti-human lipoprotein (a) monoclonal antibody of Mus or goat-anti human lipoprotein (a) monoclonal antibody in any one
Kind.
Described reducing agent includes but is not limited to dithiothreitol, DTT (DTT), beta -mercaptoethanol (ME), three (2- carboxyethyls) phosphine
(TCEP) one or more in, more preferably three (2- carboxyethyls) phosphine (TCEP).
3), in PBS (20mmol/L, PH7.0), 25 DEG C of orientations are even for the antibody after activating latex microsphere and reducing
Connection 60min, after mercaptoethanol (10mmol/L) is quenched 30min, high speed centrifugation (10000rpm, 30min) removes supernatant, uses
PBS (20mmol/L, PH7.0) is washed three times, is made into 1% (w/v) mixing with PBS (20mmol/L, PH7.0)
Liquid, 25 DEG C of concussion 30min are resuspended, obtain anti-human lipoprotein (a) latex particle.
The detection method of test kit of the present invention is latex enhancing immune turbidimetry, and test condition and parameter are as follows：
1) detecting instrument：There is 600nm wavelength, the biochemistry analyzer of 37 DEG C of thermostats.
2) sample to be tested：Fresh not haemolysis serum, can 2-8 DEG C of preservation.
3) program is specifically detected：
4) calibration procedure：Multiple spot is calibrated, using gamma correction pattern.
5) result of calculation：To determine pipe Δ A, lipoprotein (a) content can be tried to achieve according to calibration curve.
Advantage of the invention is that：Using two kinds of different-grain diameter polystyrene latex microspheres, while sensitivity is lifted,
Have preferably linearly, improve properties of product.Make antibody closure controllable using orientation coupling method, improve antibody profit
With rate, and realize efficiently being combined with sample antigens.After reducing the randomness of antibody coupling, greatly reduce to differences between batches control
Difficulty.This method can be common to various different antibodies or protein and be coupled with latex microsphere, it is easy to form the work of scale
Skill.
Description of the drawings：
Fig. 1 is the linear correlation curve figure of the present invention in embodiment 1, using AU480 automatic clinical chemistry analyzers, to 5
The reagent of gradient concentration is measured, and carries out correlation analysiss to measured value.What wherein X-axis was represented is diluted concentration, and Y-axis is represented
Be measured value average.Correlation coefficient：r2=0.9940, linear equation is：Y=1.0394x-24.5796.
Fig. 2 is the linear correlation curve figure of the present invention in embodiment 2, using AU480 automatic clinical chemistry analyzers, to 5
The reagent of gradient concentration is measured, and carries out correlation analysiss to measured value.What wherein X-axis was represented is diluted concentration, and Y-axis is represented
Be measured value average.Correlation coefficient：r2=0.9956, linear equation is：Y=1.0170x-8.9948.
Fig. 3 is the present invention and the commercial reagent box A linear correlation figure in embodiment 3, be respectively adopted test kit of the present invention with
Commercial reagent box A, using AU480 automatic clinical chemistry analyzers, to 50 parts of samples (comprising normal and exceptional sample), by each ginseng
Number is measured.Wherein what X-axis was represented is the measured value of test kit of the present invention, and what Y-axis was represented is the measure of commercial reagent box A
Value.Correlation coefficient：r2=0.9971, linear equation is：Y=1.000x+2.016.
Fig. 4 is the present invention and the commercial reagent box B linear correlation figure in embodiment 4, be respectively adopted test kit of the present invention with
Commercial reagent box B, using AU480 automatic clinical chemistry analyzers, to 50 parts of samples (comprising normal and exceptional sample), by each ginseng
Number is measured.Wherein what X-axis was represented is the measured value of test kit of the present invention, and what Y-axis was represented is the measure of commercial reagent box B
Value.Correlation coefficient：r2=0.9948, linear equation is：Y=1.001x+1.374.
Specific embodiment：
The present invention is illustrated specifically by the following examples, be only used as particular content of the present invention is described rather than limit this
Bright scope.
The preparation (1) of 1. lipoprotein (a) detection kit of embodiment
The preparation of anti-human lipoprotein (a) latex microsphere is identical with the process being previously mentioned in technical scheme.
The concrete composition of lipoprotein (a) detection kit is formulated as follows：
1) precision is determined：In same sample, continuous drawing is measured for 20 times, calculate the mean of measured value, standard deviation and
The coefficient of variation,
1 precision testing result of table
Coefficient of variation CV is generally used for the precision for weighing an assay method, and CV values are less, represent the assay method
As a result precision is better.For clinical chemistry test project, method precisions of the CV less than 5% is generally recognised as receiving
's.In table 1, less than 3%, CV values show that test kit of the present invention has excellent precision.
2) accuracy determination：Same quality-control product, replication 3 times is used to average, should with quality-control product target value relative deviation
In the range of ± 15%.
2 accuracy testing result of table
Relative deviation CB=5.31% in table 2, in the range of ± 15%, shows that test kit of the present invention has excellent standard
Exactness.
3) linear determination：Using deionized water by reagent dilutions into 5 gradient concentrations, each gradient concentration is detected 3 times, is taken
Meansigma methodss, make regression analyses to measured value and desired value, calculate r values and relative deviation (result is shown in Fig. 1, unit mg/L).
3 linear correlation detection result of table
Correlation coefficient is obtained by table 3：r2=0.9940, linear equation is：Y=1.0394x-24.5796, as a result shows
Test kit dependency of the present invention is good, with good specificity and accuracy.
4) Stability Determination：Test kit of the present invention is individually positioned in room temperature and 4 DEG C of refrigerators, with freshly prepared 900mg/
L human lipoproteins (a) standard substance substitute sample, determine 1 time every 1 month, the time required to coagulation occurs in record, the results are shown in Table 4.Knot
Fruit shows that test kit is placed in 4 DEG C of refrigerators and do not have within least more than 6 months obvious loss of activity, but should not be deposited in room temperature.
4 Detection of Stability result of table
5) specific assay：Selection carries out interference experiment measure, takes 4 parts of Quality Controls respectively, a copy of it as control, other
Three parts are separately added into 3 kinds of potential interference things：Bilirubin, triglyceride, hemoglobin, are measured with test kit of the present invention,
Unrestraint effect, shows that the test kit has good specificity, as a result as shown in table 5：
5 specific detection result of table
| Simulation chaff interference | Add concentration | Measured value | Matched group measured value | Accurately (CB%) |
| Bilirubin | 1.03mg/dl | 246mg/L | 250mg/L | - 1.6% |
| Triacylglycerol | 11.3mg/dl | 257mg/L | 250mg/L | 2.8% |
| Hemoglobin | 5.0g/L | 258mg/L | 250mg/L | 3.2% |
The preparation (2) of 2. lipoprotein (a) detection kit of embodiment
The preparation of anti-human lipoprotein (a) latex microsphere is identical with the process being previously mentioned in technical scheme.
The concrete composition of lipoprotein (a) detection kit is formulated as follows：
1) precision is determined：In same sample, continuous drawing is measured for 20 times, calculate the mean of measured value, standard deviation and
The coefficient of variation,
6 precision testing result of table
Coefficient of variation CV is generally used for the precision for weighing an assay method, and CV values are less, represent the assay method
As a result precision is better.For clinical chemistry test project, method precisions of the CV less than 5% is generally recognised as receiving
's.In table 6, less than 3%, CV values show that test kit of the present invention has excellent precision.
2) accuracy determination：Same quality-control product, replication 3 times is used to average, should with quality-control product target value relative deviation
In the range of ± 15%.
7 accuracy testing result of table
Relative deviation CB=8.65% in table 7, in the range of ± 15%, shows that test kit of the present invention has excellent standard
Exactness.
3) linear determination：Using deionized water by reagent dilutions into 5 gradient concentrations, each gradient concentration is detected 3 times, is taken
Meansigma methodss, make regression analyses to measured value and desired value, calculate r values and relative deviation (result is shown in Fig. 2, unit mg/L).
8 linear correlation detection result of table
| Diluted concentration | 150.00 | 250.00 | 500.00 | 750.00 | 1000.00 |
| Measured value | 151.35 | 256.71 | 480.58 | 721.69 | 1039.35 |
| 144.33 | 260.08 | 500.29 | 721.62 | 1032.58 | |
| 141.77 | 268.52 | 482.01 | 717.36 | 1031.84 | |
| Average | 146 | 262 | 488 | 720 | 1035 |
| Absolute deviation | 2.26 | 16.52 | 11.87 | 33.52 | 26.61 |
| Relative deviation | - 1.58% | - 6.74% | 2.38% | 4.45% | - 2.64% |
Correlation coefficient is obtained by table 8：r2=0.9956, linear equation is：Y=1.0170x-8.9948, as a result shows
Test kit dependency of the present invention is good, with good specificity and accuracy.
4) Stability Determination：Test kit of the present invention is individually positioned in room temperature and 4 DEG C of refrigerators, with freshly prepared 900mg/
L human lipoproteins (a) standard substance substitute sample, determine 1 time every 1 month, the time required to coagulation occurs in record, the results are shown in Table 9.Knot
Fruit shows that test kit is placed in 4 DEG C of refrigerators and do not have within least more than 6 months obvious loss of activity, but should not be deposited in room temperature.
9 Detection of Stability result of table
| Under room temperature | There is the time (min) of coagulation | At 4 DEG C | There is the time (min) of coagulation |
| New preparation | 1 | New preparation | 1 |
| Storage 1 month | 1 | Storage 1 month | 1 |
| Storage 2 months | 2 | Storage 2 months | 1 |
| Storage 3 months | Not coagulation | Storage 3 months | 1 |
| Storage 4 months | Not coagulation | Storage 4 months | 2 |
| Storage 5 months | Not coagulation | Storage 5 months | 2 |
| Storage 6 months | Not coagulation | Storage 6 months | 3 |
5) specific assay：Selection carries out interference experiment measure, takes 4 parts of Quality Controls respectively, a copy of it as control, other
Three parts are separately added into 3 kinds of potential interference things：Bilirubin, triglyceride, hemoglobin, are measured with test kit of the present invention,
Unrestraint effect, shows that the test kit has good specificity, as a result as shown in table 10：
10 specific detection result of table
3. test kit of the present invention of embodiment is compared with the performance indications of commercial reagent box A：
The preparation of lipoprotein (a) detection kit of the present invention：
The preparation of anti-human lipoprotein (a) latex microsphere is identical with the process being previously mentioned in technical scheme.
The concrete composition of lipoprotein (a) detection kit is formulated as follows：
The commercial reagent box A of control, its information are as follows：
Name of product：LP(a) (Lpa) determines test kit (Shanghai Ju Chuan Pharmaceutical Technology Co., Ltd)
Method：Latex strengthens turbidimetry
Dosage form：Liquid double reagent (4: 1)
Reagent components：
Analysis method：6 points of standard curve scaling methods, i.e. reagent R1, reagent R2 consumptions are respectively 240 μ l and 60 μ l, sample
4 μ l of consumption.240 μ l reagents R1 add 4 μ l samples, after 37 DEG C of reaction 5min add 60 μ l reagent R2, are determining wavelength 600nm
Under, read each 1 reading of pipe absorbance A immediately, after 37 DEG C of reaction 5min, read each 2 reading of pipe absorbance A, Δ A=A2-
A1.
Detecting instrument：AU480
Commercial reagent box A by specification operations.
1) precision is determined：In same sample, continuous drawing is measured for 20 times, calculate the mean of measured value, standard deviation and
The coefficient of variation,
11 precision testing result of table
Coefficient of variation CV is generally used for the precision for weighing an assay method, and CV values are less, represent the assay method
As a result precision is better.For clinical chemistry test project, method precisions of the CV less than 5% is generally recognised as receiving
's.In table 11, less than 5%, CV values show that test kit of the present invention equally has preferable precision with commercial reagent box A.
2) accuracy determination：Same quality-control product, replication 3 times is used to average, should with quality-control product target value relative deviation
In the range of ± 15%.
12 accuracy testing result of table
Test kit relative deviation CB=4.23% of the present invention in table 12, in the range of ± 15%, shows reagent of the present invention
Box has more preferable accuracy compared with commercial reagent box A.
3) linear determination：Test kit of the present invention and commercial reagent box A is respectively adopted, is analyzed using AU480 full-automatic biochemicals
Instrument, to 50 parts of samples (comprising normal and exceptional sample), is measured by each autoregressive parameter, and carries out correlation analysiss to measured value
(result is shown in Fig. 3, and what X-axis was represented is the measured value of test kit of the present invention, and what Y-axis was represented is the measured value of commercial reagent box A).Phase
Relation number：r2=0.9971, linear equation is：Y=1.000x+2.016, as a result shows test kit of the present invention and commercial reagent box
A dependencys are good.
13 linear correlation detection result of table
4. detection kit of the present invention of embodiment is compared with the performance indications of commercial reagent box B：
The preparation of lipoprotein (a) detection kit of the present invention：
The preparation of anti-human lipoprotein (a) latex microsphere is identical with the process being previously mentioned in technical scheme.
The concrete composition of lipoprotein (a) detection kit is formulated as follows：
The commercial reagent box B of control, its information are as follows：
Name of product：LP(a) detection kit (Japan's consonance)
Method：Latex turbidimetry
Dosage form：Liquid double reagent (4: 1)
Reagent components：
Reagent 1：Buffer
Reagent 2：Anti-human Lp (a) (goat) antibody toleration latex
Analysis method：Multiple spot is calibrated, i.e. reagent R1, and reagent R2 consumptions are respectively 280 μ l and 70 μ l, 3 μ l of sample consumption.
280 μ l reagents R1 add 3 μ l samples, add 70 μ l reagent R2, in the case where wavelength 600nm is determined, immediately after 37 DEG C of reaction 5min
Each 1 reading of pipe absorbance A is read, after 37 DEG C of reaction 10min, each 2 reading of pipe absorbance A, Δ A=A2-A1 is read.
Detecting instrument：AU480
Commercial reagent box B by specification operations.
1) precision is determined：In same sample, continuous drawing is measured for 20 times, calculate the mean of measured value, standard deviation and
The coefficient of variation,
14 precision testing result of table
Coefficient of variation CV is generally used for the precision for weighing an assay method, and CV values are less, represent the assay method
As a result precision is better.For clinical chemistry test project, method precisions of the CV less than 5% is generally recognised as receiving
's.In table 14, less than 5%, CV values show that test kit of the present invention equally has preferable precision with commercial reagent box B.
2) accuracy determination：Same quality-control product, replication 3 times is used to average, should with quality-control product target value relative deviation
In the range of ± 15%.
15 accuracy testing result of table
Test kit relative deviation CB=3.56% of the present invention in table 15, in the range of ± 15%, shows reagent of the present invention
Box equally has preferable accuracy with commercial reagent box B.
3) linear determination：Test kit of the present invention and commercial reagent box B is respectively adopted, is analyzed using AU480 full-automatic biochemicals
Instrument, to 50 parts of samples (comprising normal and exceptional sample), is measured by each autoregressive parameter, and carries out correlation analysiss to measured value
(result is shown in Fig. 4, and what X-axis was represented is the measured value of test kit of the present invention, and what Y-axis was represented is the measured value of commercial reagent box B).Phase
Relation number：r2=0.9948, linear equation is：Y=1.001x+1.374, as a result shows test kit of the present invention and commercial reagent box
B dependencys are good.
16 linear correlation detection result of table
Claims (14)
1. a kind of test kit of latex orientation coupling technology detection lipoprotein (a), based on latex enhancing immune turbidimetry, transports
Coupling technology is oriented with antibody, two kinds is activated respectively by N-hydroxy-succinamide/maleimide heterobifunctional crosslinker
The aminopolystyrene latex microsphere of particle diameter, reducing process reduce anti-human lipoprotein (a) monoclonal antibody, make two kinds of grains after activation
The latex microsphere in footpath is coupled with anti-human lipoprotein (a) the monoclonal antibody orientation after reduction respectively, forms anti-human lipoprotein (a) glue
Newborn granule.
2. antibody according to claim 1 orients coupling technology, it is characterised in that with N-hydroxy-succinamide/maleoyl
Activator of the imines heterobifunctional crosslinker as the aminopolystyrene latex microsphere of two kinds of particle diameters, forms activated state latex micro-
Ball；Reducing process reduces anti-human lipoprotein (a) monoclonal antibody, makes antibody disulfide bond be unfolded into free sulfhydryl groups, with activated state glue
Newborn microsphere is coupled, and forms stable thioether bond, so as to realize that orientation is coupled.
3. N-hydroxy-succinamide according to claim 1/maleimide heterobifunctional crosslinker, it is characterised in that
Including but not limited to 4- (N- maleimidomehyls) hexamethylene -1- carboxylic acid sulfonic group butanimide ester sodium salt (Sulfo-
SMCC), 4- (N- maleimidomehyls) hexamethylene -1- carboxylic acids sulfonic group succinimide ester (SMCC), sulfosuccinic acyl are sub-
Amido 4- (p- maleimide phenyls) butyl ester (Sulfo-SMPB), succinimido 4- (p- maleimide phenyls) butanoic acid
Salt (SMPB), succinimido -6- [(β-maleimide propionamido-)] own ester (SMPH), m- maleimide benzene first
Acyl-N- hydroxy thiosuccinimide esters (Sulfo-MBS), N- [κ-maleimide undecanoyl oxygen]-thiosuccimide
Ester (Sulfo-KMUS), N- [γ-maleimide butyryl oxygen] succinimide ester (Sulfo-GMBS), N- [ε-maleimide
Amine acetyl group oxygen] one or more in thiosuccimide ester (Sulfo-EMCS), more preferably 4- (N- maleimide first
Base) hexamethylene -1- carboxylic acids sulfonic group butanimide ester sodium salt (Sulfo-SMCC).
4. the aminopolystyrene latex microsphere of two kinds of particle diameters according to claim 1, it is characterised in that particle diameter is selected respectively
100-200nm is selected, two kinds of specifications of 200-300nm, more preferably particle diameter are respectively 110nm and 200nm.Particle fraction is 20: 80-80
: 20, more preferably 60: 40.
5. anti-human lipoprotein (a) latex microsphere is prepared as follows：
1) activation of aminopolystyrene latex microsphere
The aminopolystyrene latex microsphere for taking two kinds of particle diameters that concentration is 2.5% (w/v) respectively is sub- with N- hydroxysuccinimidyls acyl
Amine/maleimide heterobifunctional crosslinker, by rate of charge for 1: 5 (weight ratio) be mixed in PBS (20mmol/L,
PH7.0, in), 25 DEG C of activation 30-60min, high speed centrifugation (10000rpm, 30min) remove supernatant, use PBS
(20mmol/L, PH7.0) washes twice the unnecessary cross-linking agent of removal, is made into PBS (20mmol/L, PH7.0) after washing
1% (w/v) mixed liquor, 25 DEG C of concussion 10-20min are resuspended, obtain activation latex microsphere.
2) anti-human lipoprotein (a) monoclonal antibody reduction
Anti-human lipoprotein (a) monoclonal antibody and reducing agent (1-10mM) is taken, is mixed in for 1: 800 (weight ratio) by rate of charge
In PBS (20mmol/L, PH7.0), 25 DEG C of reduction 30min remove unnecessary going back by 200 chromatography of superdex
Former agent, adds iodoacetamide 0.5mg, 37 DEG C of closing sulfydryl 30-45min.
3) in PBS (20mmol/L, PH7.0), 25 DEG C of orientations are coupled the antibody after activating latex microsphere and reducing
60min, after mercaptoethanol (10mmol/L) is quenched 30min, high speed centrifugation (10000rpm, 30min) removes supernatant, uses PBS
Buffer (20mmol/L, PH7.0) is washed three times, is made into 1% (w/v) mixed liquor with PBS (20mmol/L, PH7.0),
25 DEG C of concussion 30min are resuspended, obtain anti-human lipoprotein (a) latex particle.
6. according to step 2 in claim 5) described in anti-human lipoprotein (a) monoclonal antibody, it is characterised in that for the anti-human fat of Mus
Any one in albumen (a) monoclonal antibody or goat-anti human lipoprotein (a) monoclonal antibody.
7. according to step 2 in claim 5) described in reducing agent, it is characterised in that including but not limited to dithiothreitol, DTT
(DTT), beta -mercaptoethanol (ME), one or more in three (2- carboxyethyls) phosphine (TCEP), more preferably three (2- carboxyethyls) phosphine
(TCEP).
8. test kit of the present invention includes reagent 1 and reagent 2, and reagent 1 is 4: 1 with reagent 2.Reagent 1 include buffer, stabilizer and
Preservative, reagent 2 include anti-human lipoprotein (a) latex microsphere, buffer, protective agent, stabilizer and preservative.
9. the buffer in reagent according to claim 81 and reagent 2, it is characterised in that including but not limited to Tris-
HCL buffer, PBS, disodium hydrogen phosphate-citrate buffer solution, the one kind in potassium dihydrogen phosphate-sodium hydrate buffer solution
Or several, more preferably PBS.PH scopes are 6.5-8.5, more preferably PH6.5-7.5.
10. the stabilizer in reagent according to claim 81, it is characterised in that including but not limited to Macrogol 8000,
Sucrose, glycerol, glucose, gelatin, sodium chloride, potassium chloride, sodium carbonate, sodium sulfate, one or more in potassium sulfate, more preferably
Macrogol 8000.
Protective agent in 11. reagents according to claim 82 is EDTA.
Stabilizer in 12. reagents according to claim 82, it is characterised in that including but not limited to Macrogol 8000,
Sucrose, glycerol, glucose, gelatin, sodium chloride, potassium chloride, sodium carbonate, sodium sulfate, one or more in potassium sulfate, more preferably
Sucrose.
Preservative in 13. reagents according to claim 81 and reagent 2, it is characterised in that including but not limited to nitrine
Sodium, proclin 300, one or more in thimerosal.
14. lipoprotein (a) detection kit of the present invention, concentration is
Reagent R1：PBS (PH7.0) 20～100mmol/L
Macrogol 8000 (PEG 8000) 10～30g/L
Sodium azide 0.05%
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| CN113049811A (en) * | 2021-03-22 | 2021-06-29 | 深圳市亚辉龙生物科技股份有限公司 | Nano magnetic bead coating material, preparation method thereof, detection reagent and detection kit |
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Application publication date: 20170315 |