CN106526198A - Combined orientation agents optimized latex coupled antibody detection method of prealbumin (PA) - Google Patents
Combined orientation agents optimized latex coupled antibody detection method of prealbumin (PA) Download PDFInfo
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- G—PHYSICS
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- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The invention provides a combined orientation agents optimized latex coupled antibody detection method of prealbumin (PA). The method is based on a latex enhanced turbidimetric immunoassay method, and is characterized in that combined orientation agents are utilized: an orientation agent 1 and an orientation agent 2. Firstly, the orientation agent 1 is combined with a PA antibody to form a PA antibody compound; the orientation agent 2 is then combined with the PA antibody compound; and finally, the compound is respectively coupled with carboxyl latex microspheres of two different particle sizes to form PA latex particles. By the method, the formed reagent has high sensitivity and good precision and linearity, is simple to prepare and is worthy of further promotion and application.
Description
Technical field：
The present invention relates to medical science detection and in-vitro diagnosis field, before a kind of combination directing agent optimization latex coupled antibody detection
The method of albumin (PA).
Background technology：
Prealbumin (Prealbumin, PA), also known as transthyretin (transthyretin), molecular weight 5.4
Ten thousand, synthesized by hepatocyte, in electrophoretic separation, be often displayed in albuminous front, its half-life is very short, only about 1.9 days.Cause
This, determine its concentration in blood plasma for understanding the malnutrition of protein, hepatic insufficiency, than albumin and turn ferrum egg
There is higher sensitivity in vain.Except used as a kind of sensitive nutrient protein index, PA is in acute inflammation, malignant tumor, liver
When hardening or nephritis, its blood concentration declines.
Clinic there is no the reference method that generally acknowledged blood-serum P A is determined, the method that clinical laboratory determines blood-serum P A content at present
Mainly there is immunoturbidimetry, including Immune scatter turbidimetry and Immunity transmission turbidity.In recent years, turbidimetry for Determination technology is because of which
With it is easy, fast and automatically, it is micro the features such as, be increasingly becoming the conventional method that clinical laboratory determines blood-serum P A, and due to glue
The rise and fast development of newborn microsphere coupling technology, makes immunoturbidimetry be combined with latex microsphere gradually, defines common immunity ratio
Deriving for turbid technology, becomes a kind of easier compared to immunoturbidimetry quick, and sensitivity is higher, the higher detection side of signal
Method, but the method there is also its limitation, and the uncontrollability of coupling effect so that and the utilization rate of latex microsphere is not high, therefore,
A kind of method for lifting coupling effect between antibody and latex microsphere is found, the utilization rate of latex microsphere, intensified response effect is improved
It is the technical problem of this area urgent need to resolve.
The content of the invention：
A kind of in view of above technical problem, it is an object of the invention to provide combination directing agent optimization latex coupled antibody inspection
The method for surveying prealbumin (PA), described method are based on latex enhancing immune turbidimetry, and its feature is using a kind of combination
Directing agent：Directing agent 1 and directing agent 2, first combine PA antibody according to directing agent 1, form PA antibody complexes, and directing agent 2 is tied again
PA antibody complexes are closed, finally is coupled to form PA latex microsphere granules with the carboxylated latex microsphere of two kinds of different-grain diameters respectively.
Technical scheme：By the directing agent 1 in combination directing agent and PA antibodies, form PA antibody and be combined
Thing.By the directing agent 2 in combination directing agent and PA antibody complexes in conjunction with.It is eventually adding the activated of two kinds of different-grain diameters
Carboxylated latex microsphere is coupled, and forms PA latex particles.Detection kit is prepared based in this approach, and reagent 1 includes delaying
Liquid, stabilizer and preservative is rushed, reagent 2 includes PA latex microspheres, buffer, protective agent, suspending agent, stabilizer and preservative.
Directing agent 1 described in the technical program is N- hydroxysuccinimide esters (BNHS) or long-armed activated biotin
(BCNHS) it is, more preferably long-armed activated biotin (BCNHS).Described directing agent 2 is Streptavidin.
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, barbital sodium-hydrochloride buffer, the one kind in potassium dihydrogen phosphate-sodium hydrate buffer solution, boric acid-borate buffer solution
Or several, more preferably PBS.PH scopes are 7.0-8.0, more preferably PH7.5-8.0.
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, Macrogol 8000
With Sodium Chloride, concentration ratio is 2: 1.
Preservative in reagent 1 described in test kit of the present invention and reagent 2 includes but is not limited to sodium azide, proclin300,
One or more in thimerosal.
Protective agent in reagent 2 described in test kit of the present invention is EDTA.
Suspending agent in reagent 2 described in test kit of the present invention includes but is not limited to ethylene glycol, glycerol, maltose, sucrose
In one or more, more preferably glycerol.
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.
In the present invention, the preparation of preparation and PA detection kit including PA latex particles.Concrete operations are as follows：
The preparation of PA latex particles：
1) directing agent 1 combines PA antibody
It is 0.5-5 μ g/ml goat-anti people's prealbumin monoclonal antibodies to take concentration, with directing agent 1：Long-armed activated biotin
(BCNHS) amount ratio is 1: 2 (mg: mg), is mixed in PBS (200mmol/L, PH7.5), by BCNHS and dimethyl
Methanamide (DMF) rate of charge is that 40: 1 (w/v) add DMF, after 37 DEG C of constant temperature oscillation reaction 60min, by superdex 200
Chromatography removes unnecessary BCNHS, and PBS (200mmol/L, PH7.5), 37 DEG C of constant temperature oscillation reaction 20min are resuspended, obtain
Obtain PA antibody complexes.
2) directing agent 2 combines PA antibody complexes
By directing agent 2：Streptavidin is mixed in PBS with the rate of charge of PA antibody complexes for 1: 2 (w/v)
In (200mmol/L, PH7.5), 37 DEG C of constant temperature oscillations react 30min, remove unnecessary chain by 200 chromatography of superdex
Mould Avidin, PBS (200mmol/L, PH7.5), 37 DEG C of constant temperature oscillation reaction 30min are resuspended, and acquisition Streptavidin-
PA antibody complexes.
3) activation of latex microsphere
Take the carboxyl polystyrene that particle diameter is respectively 50nm-100nm (2.5%w/v) and 200nm-300nm (5.0%w/v)
Latex microsphere, after being washed with PBS (200mmol/L, PH7.5) for 80: 20 respectively by particle fraction, is redissolved in PBS
In buffer (200mmol/L, PH7.5), 1% (w/v) carbodiimides (EDC) activation is separately added into, 37 DEG C of constant temperature oscillations are anti-
20min is answered, is centrifuged (12000rpm, 30min), removed supernatant, washed twice with PBS (200mmol/L, PH7.5)
Afterwards, in being dissolved in PBS (200mmol/L, PH7.5) respectively, 37 DEG C of constant temperature oscillation reaction 30min are resuspended, obtain the two of activation
The latex microsphere of particle diameter is planted,.
4) Streptavidin-PA antibody complexes are coupled with latex microsphere
By step 2) Streptavidin-PA antibody complexes that obtain are added separately to step 3 by 3-5: 1 volume ratio)
In the activation latex microsphere of the two kinds of particle diameters for obtaining, 37 DEG C of constant temperature oscillations react 2h, close 20min with 0.5% (w/v) lysine
Afterwards, high speed centrifugation (12000rpm, 30min) is carried out respectively, is removed supernatant, is washed with PBS (200mmol/L, PH7.5)
Wash three times, in being dissolved in PBS (200mmol/L, PH7.5) respectively, 37 DEG C of constant temperature oscillation reaction 30min are resuspended, finally by two
Plant the PA latex microspheres granule mixing of particle diameter.
The preparation of PA detection kit：
With above-mentioned preferred material reagent preparation box, (liquid double reagent, R1: R2 is 3: 1), concentration is respectively
The test kit prepared based in the process of the present invention, its detection method are latex enhancing immune turbidimetry, are tested
Condition and parameter are as follows：
1) detecting instrument：Biochemistry analyzer with 600nm wavelength, 37 DEG C of thermostats.
2) sample to be tested：Fresh not haemolysis serum, can 2-8 DEG C of preservation.
3) specifically detect program：
4) use of calibration object and curve plotting method：Redissolved with 2.0ml distilled water, calibration solution is carried out again with distilled water
Than dilution, calibration mode is calibrated using multiple spot.
5) result of calculation
PA contents (mg/L)=Δ AT/ Δ AS × calibration solution concentration in sample
In formula：
ΔAT：With blank tube absorbance as the sample cell absorbance of control
ΔAS：With blank tube absorbance as the calibration pipe absorbance of control
Advantage of the invention is that：Using two kinds of different-grain diameter carboxyl polystyrene latex microspheres, sensitivity is being lifted
Meanwhile, with preferably linear, improve properties of product.Improved using combination directing agent and be coupled between antibody and latex microsphere effect
Really, the utilization rate of latex microsphere, intensified response effect are improve.
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=0.9931, linear equation is：Y=0.9932x-0.3201.
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=0.9952, linear equation is：Y=0.9941x+1.5590.
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=0.9940, linear equation is：Y=0.996x+2.497.
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=0.9930, linear equation is：Y=1.007x+0.513.
Specific embodiment：
The present invention is specifically illustrated by the following examples, is only made particular content to illustrate the invention rather than is limited this
Bright scope.
The preparation (1) of embodiment 1. prealbumin (PA) detection kit
The preparation of PA latex particles is identical with the process being previously mentioned in technical scheme.
The concrete composition of prealbumin (PA) 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 weighing the precision of 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, CV values are less than 5%, 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=5.32% 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
| Diluted concentration | 90.00 | 150.00 | 300.00 | 450.00 | 600.00 |
| Measured value | 85.67 | 155.48 | 308.66 | 430.73 | 595.86 |
| 82.62 | 151.40 | 321.38 | 416.25 | 624.90 | |
| 83.78 | 155.26 | 306.23 | 409.11 | 605.38 | |
| Average | 84 | 154 | 312 | 419 | 609 |
| Absolute deviation | 5.04 | 5.39 | 14.45 | 27.92 | 13.12 |
| Relative deviation | 5.66% | - 3.62% | - 4.86% | 6.25% | - 2.20% |
Correlation coefficient is obtained by table 3：r2=0.9931, linear equation is：Y=0.9932x-0.3201, 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 into room temperature and 4 DEG C of refrigerators, with freshly prepared 300mg/
L prealbumins 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.As a result
Show, 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
| Under room temperature | There is the time (min) of coagulation | At 4 DEG C | There is the time (min) of coagulation |
| It is new to prepare | 0.5 | It is new to prepare | 0.5 |
| Storage 1 month | 2 | Storage 1 month | 0.5 |
| Storage 2 months | 3 | Storage 2 months | 1 |
| Storage 3 months | 8 | Storage 3 months | 2 |
| 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 | 2 |
5) specific assay：Selection carries out interference experiment measure, takes 5 parts of Quality Controls respectively, a copy of it as control, other
Three parts are separately added into 4 kinds of potential interference things：Bilirubin, triglyceride, hemoglobin, rheumatoid factor, use test kit of the present invention
It is measured, equal 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 | 342μmol/L | 296mg/L | 300mg/L | - 1.3% |
| Triglyceride | 8.7mmol/L | 297mg/L | 300mg/L | - 1.0% |
| Hemoglobin | 5.0g/L | 288mg/L | 300mg/L | - 4.0% |
| Rheumatoid factor | 75U/L | 307mg/L | 300mg/L | 2.3% |
The preparation (2) of embodiment 2. prealbumin (PA) detection kit
The preparation of PA latex particles is identical with the process being previously mentioned in technical scheme.
The concrete composition of prealbumin (PA) 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 weighing the precision of 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, CV values are less than 5%, 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=8.68% 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 | 90.00 | 150.00 | 300.00 | 450.00 | 600.00 |
| Measured value | 87.43 | 142.89 | 312.19 | 434.63 | 600.10 |
| 82.69 | 145.74 | 323.77 | 435.32 | 586.58 | |
| 80.95 | 143.72 | 340.97 | 455.60 | 592.74 | |
| Average | 84 | 144 | 326 | 442 | 593 |
| Absolute deviation | 7.34 | 6.56 | 25.85 | 7.06 | 4.89 |
| Relative deviation | 8.06% | 4.35% | - 8.62% | 1.57% | 0.82% |
Correlation coefficient is obtained by table 8：r2=0.9952, linear equation is：Y=0.9941x+1.5590, 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 into room temperature and 4 DEG C of refrigerators, with freshly prepared 300mg/
L prealbumins 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.As a result
Show, 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
5) specific assay：Selection carries out interference experiment measure, takes 5 parts of Quality Controls respectively, a copy of it as control, other
Three parts are separately added into 4 kinds of potential interference things：Bilirubin, triglyceride, hemoglobin, rheumatoid factor, use test kit of the present invention
It is measured, equal unrestraint effect shows that the test kit has good specificity, as a result as shown in table 10：
10 specific detection result of table
| Simulation chaff interference | Add concentration | Measured value | Matched group measured value | Accurately (CB%) |
| Bilirubin | 342μmol/L | 289mg/L | 300mg/L | - 3.7% |
| Triglyceride | 8.7mmol/L | 305mg/L | 300mg/L | 1.7% |
| Hemoglobin | 5.0g/L | 309mg/L | 300mg/L | 3.0% |
| Rheumatoid factor | 75U/L | 291mg/L | 300mg/L | - 3.0% |
3. test kit of the present invention of embodiment is compared with the performance indications of commercial reagent box A：
The preparation of prealbumin (PA) detection kit：
The preparation of PA latex particles is identical with the process being previously mentioned in technical scheme.
The concrete composition of prealbumin (PA) detection kit is formulated as follows：
The commercial reagent box A of control, its information are as follows：
Name of product：PA detection test kit (Sichuan mikey science and technology limited Company)
Method：Immunity transmission turbidity
Dosage form：Liquid single agents (R：2×35ml)
Kit forms：
R：
PA reagents
Goat-anti people's PA serum
Disappear fat agent
STD：
Prealbumin calibrates serum
Basic parameter：
Method：End-point method；Sample/reagent：1/100；Dominant wavelength：340nm；Commplementary wave length：700nm；Reaction temperature：37℃；
Response time：10min.Determination step：
Calculate：Using multiple spot it is non-linear/spline function calibration mode automatically generates determination sample after calibration curve by instrument
Content or using each standard pipe concentration value as abscissa, absorbance change value is looked into as vertical coordinate, manual drawing canonical plotting
Sample size is obtained by readding the figure.
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 weighing the precision of 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, CV values are less than 5%, show that test kit of the present invention has more preferable precision than 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=4.20% 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=0.9940, linear equation is：Y=0.996x+2.497, 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. test kit of the present invention of embodiment is compared with the performance indications of commercial reagent box B：
The preparation of prealbumin (PA) detection kit：
The preparation of PA latex particles is identical with the process being previously mentioned in technical scheme.
The concrete composition of prealbumin (PA) detection kit is formulated as follows：
The commercial reagent box B of control, its information are as follows：
Name of product：PA detection test kit (Siemens of the U.S.)
Method：Immunoturbidimetry
Dosage form：Liquid single agents (R：4×30ml)
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 weighing the precision of 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, CV values are less than 5%, 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 has more preferable accuracy compared 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=0.9930, linear equation is：Y=1.007x+0.513, 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 (6)
1. a kind of method that combination directing agent optimization latex coupled antibody detects prealbumin (PA), described method are based on latex
Strengthen immunoturbidimetry, its feature is using a kind of combination directing agent：Directing agent 1 and directing agent 2, first tie according to directing agent 1
Close PA antibody, form PA antibody complexes, directing agent 2 in conjunction with PA antibody complexes, finally respectively with two kinds of different-grain diameters
Carboxylated latex microsphere is coupled to form PA latex particles.
2. combination directing agent according to claim 1：Directing agent 1 and directing agent 2, it is characterised in that directing agent 1 is N- hydroxyls
Base succimide ester (BNHS) or long-armed activated biotin (BCNHS), it is more preferably long-armed activated biotin (BCNHS).It is described
Directing agent 2 be Streptavidin.
3. the carboxylated latex microsphere of two kinds of different-grain diameters according to claim 1, it is characterised in that particle diameter is respectively 50nm-
100nm (2.5%w/v) and 200nm-300nm (5.0%w/v), particle fraction is 80: 20.
4. in the process of the present invention based on prepare detection kit, reagent 1 includes buffer, stabilizer and preservative, and reagent 2 is wrapped
Include PA latex microspheres, buffer, protective agent, suspending agent, stabilizer and preservative.Buffer bag in the reagent 1 and reagent 2
Tris-HCL buffer is included but is not limited to, PBS, barbital sodium-hydrochloride buffer, potassium dihydrogen phosphate-sodium hydroxide delay
Rush one or more in liquid, boric acid-borate buffer solution, more preferably PBS.PH scopes are 7.0-8.0, more preferably
PH7.5-8.0.Stabilizer in the reagent 1 includes but is 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 and Sodium Chloride,
Concentration ratio is 2: 1.The reagent 1 includes but is not limited to sodium azide with the preservative in reagent 2, and proclin 300, in thimerosal
One or more.Protective agent in the reagent 2 is EDTA.Suspending agent in the reagent 2 includes but is not limited to ethylene glycol,
Glycerol, maltose, more preferably one or more in sucrose, glycerol.Stabilizer in the reagent 2 is included but is not limited to
Macrogol 8000, sucrose, glycerol, glucose, gelatin, Sodium Chloride, potassium chloride, sodium carbonate, sodium sulfate, the one kind in potassium sulfate
Or several, more preferably sucrose.
The preparation of 5.PA latex particles：
1) directing agent 1 combines PA antibody
Concentration is taken for 0.5-5 μ g/ml goat-anti people's prealbumin monoclonal antibodies, and directing agent 1: long-armed activated biotin
(BCNHS) amount ratio is 1: 2 (mg: mg), is mixed in PBS (200mmol/L, PH7.5), by BCNHS and dimethyl
Methanamide (DMF) rate of charge is that 40: 1 (w/v) add DMF, after 37 DEG C of constant temperature oscillation reaction 60min, by superdex 200
Chromatography removes unnecessary BCNHS, and PBS (200mmol/L, PH7.5), 37 DEG C of constant temperature oscillation reaction 20min are resuspended, obtain
Obtain PA antibody complexes.
2) directing agent 2 combines PA antibody complexes
By directing agent 2: Streptavidin is mixed in PBS with the rate of charge of PA antibody complexes for 1: 2 (w/v)
In (200mmol/L, PH7.5), 37 DEG C of constant temperature oscillations react 30min, remove unnecessary chain by 200 chromatography of superdex
Mould Avidin, PBS (200mmol/L, PH7.5), 37 DEG C of constant temperature oscillation reaction 30min are resuspended, and acquisition Streptavidin-
PA antibody complexes.
3) activation of latex microsphere
Take the carboxyl polystyrene latex that particle diameter is respectively 50nm-100nm (2.5%w/v) and 200nm-300nm (5.0%w/v)
Microsphere, after being washed with PBS (200mmol/L, PH7.5) for 80: 20 respectively by particle fraction, is redissolved in PBS bufferings
In liquid (200mmol/L, PH7.5), 1% (w/v) carbodiimides (EDC) activation, 37 DEG C of constant temperature oscillation reactions are separately added into
20min, is centrifuged (12000rpm, 30min), removes supernatant, after being washed twice with PBS (200mmol/L, PH7.5),
In being dissolved in PBS (200mmol/L, PH7.5) respectively, 37 DEG C of constant temperature oscillation reaction 30min are resuspended, obtain two kinds of activation
The latex microsphere of particle diameter,.
4) Streptavidin-PA antibody complexes are coupled with latex microsphere
By step 2) Streptavidin-PA antibody complexes that obtain are added separately to step 3 by 3-5: 1 volume ratio) obtain
Two kinds of particle diameters activation latex microsphere in, 37 DEG C of constant temperature oscillations react 2h, with 0.5% (w/v) lysine closing 20min after,
High speed centrifugation (12000rpm, 30min) is carried out respectively, supernatant is removed, and is washed with PBS (200mmol/L, PH7.5)
Three times, in being dissolved in PBS (200mmol/L, PH7.5) respectively, 37 DEG C of constant temperature oscillation reaction 30min are resuspended, finally by two kinds
The PA latex microspheres granule mixing of particle diameter.
6. PA detection kit prepared in the process of the present invention, concentration is：
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107490697A (en) * | 2017-08-10 | 2017-12-19 | 迈克生物股份有限公司 | A kind of kit for testing prealbumin and detection method |
| CN109541241A (en) * | 2019-01-25 | 2019-03-29 | 浙江夸克生物科技有限公司 | A kind of assay kit of lipoprotein (a) |
| CN109957003A (en) * | 2019-04-15 | 2019-07-02 | 南京立顶生物科技有限公司 | A kind of stable SAA mutant and its application in disease detection |
| CN110514848A (en) * | 2019-08-21 | 2019-11-29 | 深圳上泰生物工程有限公司 | A kind of glycosylated hemoglobin antibody complex and glycosylated hemoglobin detection kit |
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| CN102590524A (en) * | 2011-12-30 | 2012-07-18 | 北京九强生物技术股份有限公司 | Assay kit for neutrophil gelatinase-associated lipocalin |
| CN102759631A (en) * | 2012-08-02 | 2012-10-31 | 南京诺尔曼生物技术有限公司 | Latex enhanced turbidimetric immunoassay kit of quantitatively detecting procalcitonin PCT |
| CN102788881A (en) * | 2012-08-16 | 2012-11-21 | 北京恩济和生物科技有限公司 | Prealbumin detection reagent kit and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102590524A (en) * | 2011-12-30 | 2012-07-18 | 北京九强生物技术股份有限公司 | Assay kit for neutrophil gelatinase-associated lipocalin |
| CN102759631A (en) * | 2012-08-02 | 2012-10-31 | 南京诺尔曼生物技术有限公司 | Latex enhanced turbidimetric immunoassay kit of quantitatively detecting procalcitonin PCT |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107490697A (en) * | 2017-08-10 | 2017-12-19 | 迈克生物股份有限公司 | A kind of kit for testing prealbumin and detection method |
| CN107490697B (en) * | 2017-08-10 | 2018-10-12 | 迈克生物股份有限公司 | A kind of kit for testing prealbumin and detection method |
| CN109541241A (en) * | 2019-01-25 | 2019-03-29 | 浙江夸克生物科技有限公司 | A kind of assay kit of lipoprotein (a) |
| CN109957003A (en) * | 2019-04-15 | 2019-07-02 | 南京立顶生物科技有限公司 | A kind of stable SAA mutant and its application in disease detection |
| CN110514848A (en) * | 2019-08-21 | 2019-11-29 | 深圳上泰生物工程有限公司 | A kind of glycosylated hemoglobin antibody complex and glycosylated hemoglobin detection kit |
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