CN108490186A - A kind of kit of detection cardic fatty acid binding protein - Google Patents
A kind of kit of detection cardic fatty acid binding protein Download PDFInfo
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- CN108490186A CN108490186A CN201810158547.2A CN201810158547A CN108490186A CN 108490186 A CN108490186 A CN 108490186A CN 201810158547 A CN201810158547 A CN 201810158547A CN 108490186 A CN108490186 A CN 108490186A
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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- 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
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Abstract
The invention discloses a kind of kits of detection cardic fatty acid binding protein, the kit principle is based on latex enhancing immune turbidimetry, its technical solution is that it contains h-FABP antibody fragment compounded latex particles, h-FABP antibody is first cut into 2 segments of F (ab') by h-FABP antibody fragment compounded latexs particle by ficin, 2 segments of F (ab') are crosslinking on carboxylated latex microballoon again, form h-FABP antibody fragment compounded latex particles, to significantly reduce the non-specific binding of Fc, realization is efficiently combined with sample antigens, in 37 DEG C of constant temperature, wavelength is under 520nm, measure absorbance, calculate sample cardic fatty acid binding protein (h-FABP) content.Compared with conventional method, this kit antibody utilization rate is high, and accuracy is high, can effectively control difference between batch, and further genralrlization is worth to use.
Description
Technical field
The present invention relates to medical detection reagent box fields, and in particular to a kind of detection cardic fatty acid binding protein (h-
FABP kit).
Background technology
Cardic fatty acid binding protein (h-FABP) is the novel small cytoplasmic protein of one kind being rich in heart.By 132 ammonia
Base acid forms, molecular weight 15kDa.It has height heartspecific, is primarily present in cardiac muscle cell, but other than heart
Tissue in also have low concentration expression, as there are micro in skeletal muscle.H-FABP accounts for about in heart whole soluble protein
4%~8%.H-FABP can be combined with intramyocardial long chain fatty acids, be transported in mitochondria, final oxidation point
Solution provides energy at adenosine triphosphate atp, for cardiac activity.Therefore h-FABP is important the carrier protein of energy aliphatic acid.
Due to being free of in the blood plasma and urine of normal person or containing only denier h-FABP, when cardiac muscle cells are damaged, h-FABP is quick
It is discharged into blood and urine, is significantly increased in blood and urine in the concentration of the Super acute h-FABP of AMI.Therefore h-FABP
Concentration variation can be used as the better index and its sensibility and predictable ideal of AMI early diagnosis.
The common method of cardic fatty acid binding protein (h-FABP) detection technique has following several currently on the market：It puts
Penetrate immunoassay (RIA), fluorescence immunoassay (FIA), time-resolved fluorescent immunoassay (TRFIA), enzyme linked immunological
Measuring method (ELISA), Immunosensors Technology, immune colloidal gold technique, latex enhancing immune turbidimetry etc., are exempted from latex intensified
Epidemic disease turbidimetry is in the majority.Latex enhancing immune turbidimetry belongs to the deriving technology of common immunoturbidimetry technology, overcomes common immune
The very limited disadvantage of turbidimetric technic semaphore, makes antibody volume have the growth on the order of magnitude, greatly improves detectability,
But there is also huge technological difficulties, i.e. antibody to be connected to the direction on latex microsphere and be unable to control for this method so that its activity
And stability (including not change with the time and change and batch between consistency) it cannot be guaranteed that.Therefore, a kind of antibody is found
The technical issues of method being effectively coupled between latex microsphere is this field urgent need to resolve.
Invention content
For overcome the deficiencies in the prior art, the present invention provides a kind of detection cardic fatty acid binding protein (h-
FABP kit) contains h-FABP antibody fragment compound adhesives based on latex enhancing immune turbidimetry in the kit
Newborn particle, which uses antibody fragmentation technology, first by ficin by h-
FABP antibody cuts into 2 segments of F (ab'), then 2 segments of F (ab') are crosslinking on carboxylated latex microballoon, forms h-FABP antibody
Segment compounded latex particle, to significantly reduce the non-specific binding of Fc, realization is efficiently combined with sample antigens.
To achieve the goals above, the technical solution adopted by the present invention is：Kit includes being made of reagent 1 and reagent 2
Liquid double reagent component, wherein
1 ingredient of the reagent is as follows：
Phosphate buffer pH6.0-7.0 0.1-0.5mol/L
PEG 8000 9-15g/L
Sodium azide 0.01wt%
2 ingredient of the reagent is as follows：
The volume ratio of the reagent 1 and reagent 2 is 3:1.
Further setting is prepared by the general following methods of the h-FABP antibody fragment compounded latex particles：With
Antibody fragmentation technology first cuts h-FABP antibody by ficin under the buffer environment containing cysteine
It cuts, forms the peptide fragment of h-FABP antibody F (ab') 2 segment and Fc, be purified by flash through Protein A centrifugal columns, remove Fc
Peptide fragment, obtain h-FABP antibody F (ab') 2 segment of purifying, then h-FABP antibody F (ab') 2 segment is crosslinking in
On carboxylated latex microballoon, h-FABP antibody fragment compounded latex particles are formed.
Further setting is to include the following steps：
(1) h-FABP antibody is cut into 2 segments of F (ab') by ficin；
The anti-human h-FABP antibody of mouse is gone into salt plug desalination with centrifugal, buffer solution used is 0.2mol/L phosphate-buffereds
Liquid (pH6.5)；Identical phosphate buffer 20ml is taken, ficin 1mg, 1mol/L cysteine solution 1ml is added,
0.5mol/L EDTA solution 1ml, are placed in 37 DEG C, uniform dissolution 2h in 100r/min water bath with thermostatic control shaking tables；Cross Sephadex G50
Column is used in combination 0.2mol/L phosphate buffers (pH6.5) to be eluted, and it is molten to collect ficin enzyme of the acquisition containing activation
Liquid, and it is concentrated into original volume；With volume ratio 5：The anti-human h-FABP antibody of mouse after 1 mixing dialysis and activation ficin
Enzyme solutions are placed in 37 DEG C, and 3h is digested in 100r/min water bath with thermostatic control shaking tables, the activation ficin of equivalent is added after 3h again
Enzyme enzyme solutions continue to digest 3h, in triplicate；0.1mmol/L iodoacetamide 0.5ml ice baths 1h is added after enzymolysis to terminate instead
It answers；Protein A centrifugal columns are crossed, the 0.2mol/L phosphate buffers (pH6.5) containing 0.1mol/LNaCl is used in combination to be eluted,
Finally for 24 hours with ultra-pure water dialysis, it collects and obtains h-FABP antibody F (ab') 2 segment, be placed in 2-8 DEG C of preservation；
(2) 2 segments of F (ab') are crosslinking on carboxylated latex microballoon, form h-FABP antibody fragment compounded latex particles；
It is a concentration of 10% latex microsphere 1ml of 120nm to take grain size, and the phosphate buffer of 0.2mol/L, pH6.5 is added
9ml, room temperature shake 2mins, are rapidly added 120 μ l EDC aqueous solutions (50mg/ml) after mixing, and 150 μ l N- hydroxyls are added
Thiosuccimide aqueous solution (100mg/ml), is stirred at room temperature 40min, and 2-8 DEG C of centrifugation 30min (rotating speed 15000rpm) is used
0.2mol/L phosphate buffers (pH6.5) wash three times, remove supernatant, precipitation 0.2mol/L phosphate buffers
(pH6.5) 1ml ultrasounds are resuspended；0.5ml h-FABP antibody F (ab') 2 segment is taken, 0.5ml latex microsphere re-suspension liquids, room is added
Temperature stirring 1h, repeats and 0.5ml latex microsphere re-suspension liquids is added, and continues that 3h is stirred at room temperature, 400 μ l, 5% hydroxylamine hydrochloride water is added
Reagent and 200 μ l 1%BSA aqueous solutions is quenched in solution, and room temperature shakes 1h；4 DEG C of centrifugation 30min (rotating speed 15000rpm), precipitation are used
0.2mol/L phosphate buffers (pH6.5) wash three times, supernatant are removed, with 0.2mol/L phosphate buffers (pH6.5)
1ml ultrasonic disperses 30min, 2-8 DEG C is sealed.
Using the above scheme, advantage of the invention is that：Using antibody fragmentation, the non-specific knot of Fc is significantly reduced
It closes, realization is efficiently combined with sample antigens, is improved antibody utilization rate, is also further improved the accuracy of entire reagent, energy
Effectively control difference between batch.
Description of the drawings
The line of cardic fatty acid binding protein (h-FABP) detection kit obtained by 1 present invention specific implementation 1 of attached drawing
Property correlation figure；
Cardic fatty acid binding protein (h-FABP) detection kit obtained by 2 specific embodiment of the invention 2 of attached drawing with
The linearly related figure of commercial reagent box.
Specific implementation mode
The present invention illustrates that technical solution, protection scope of the present invention are not limited to above-mentioned specific implementation in conjunction with specific embodiments
Mode, persons skilled in the art according to the present disclosure, may be used other a variety of specific implementation modes and implement
The present invention's or every design structure using the present invention and thinking, simple change or change are done, both falls within the present invention's
Protection domain.
Prepare embodiment
Under the buffer environment containing cysteine, first h-FABP antibody is cut by ficin, forms F
(ab') peptide fragment of 2 segments and Fc is purified by flash through Protein A centrifugal columns, removes the peptide fragment of Fc, purified
2 segments of F (ab'), then 2 segments of F (ab') are crosslinking on carboxylated latex microballoon, form h-FABP antibody fragment compounded latexs
Particle utilizes obtained compounded latex particle preparation detection kit.
Concrete operations are as follows：
The preparation of 1.h-FABP antibody fragment compounded latex particles
H-FABP antibody selects the anti-human h-FABP antibody of mouse.By the anti-human h-FABP antibody Spin-OUT of mouseTM GT-
600, Medi it is centrifugal go buffer solution used in salt plug desalination be 0.2mol/L phosphate buffers (pH6.5).Take identical phosphoric acid
Ficin 1mg, 1mol/L cysteine solution 1ml, 0.5mol/L EDTA solution 1ml are added in salt buffer 20ml,
It is placed in 37 DEG C, uniform dissolution 2h in 100r/min water bath with thermostatic control shaking tables.Sephadex G50 columns are crossed, 0.2mol/L phosphate is used in combination
Buffer solution (pH6.5) is eluted, and is collected and is obtained the ficin enzyme solutions containing activation, and is concentrated into original volume.With body
Product ratio 5：The anti-human h-FABP antibody of mouse after 1 mixing dialysis and activation ficin enzyme solutions, are placed in 37 DEG C, 100r/
3h is digested in min water bath with thermostatic control shaking tables, the activation ficin enzyme solutions that equivalent is added after 3h again continue to digest 3h, weight
Again three times.0.1mmol/L iodoacetamide 0.5ml ice baths 1h is added after enzymolysis and terminates reaction.Protein A centrifugal columns are crossed,
It is used in combination the 0.2mol/L phosphate buffers (pH6.5) containing 0.1mol/LNaCl to be eluted, finally for 24 hours with ultra-pure water dialysis,
It collects and obtains h-FABP antibody F (ab') 2 segment, be placed in 2-8 DEG C of preservation.
It is a concentration of 10% latex microsphere 1ml of 120nm to take grain size, and 0.2mol/L phosphate buffers (pH6.5) are added
9ml, room temperature shake 2mins, are rapidly added 120 μ lEDC aqueous solutions (50mg/ml) after mixing, and 150 μ l sulfo- are added
40min, 2-8 DEG C of centrifugation 30min (rotating speed 15000), with 0.2mol/L phosphate is stirred at room temperature in NHS aqueous solutions (100mg/ml)
Buffer solution (PH6.5) washs three times, removes supernatant, precipitation 0.2mol/L phosphate buffers (pH6.5) 1ml ultrasound weights
It is outstanding.0.5ml h-FABP antibody F (ab') 2 segment is taken, 0.5ml latex microsphere re-suspension liquids are added, 1h is stirred at room temperature, repeats and adds
Enter 0.5ml latex microsphere re-suspension liquids, continue that 3h is stirred at room temperature, 400 μ l, 5% hydroxylamine hydrochloride aqueous solutions is added, reagent and 200 is quenched
μ l 1%BSA aqueous solutions, room temperature shake 1h；4 DEG C of centrifugation 30min (rotating speed 15000), precipitation 0.2mol/L phosphate buffers
(pH6.5) washing three times, removes supernatant, with 0.2mol/L phosphate buffers (pH6.5) 1ml ultrasonic disperses 30min, 2-8
It DEG C is sealed.
The preparation of cardic fatty acid binding protein 2. (h-FABP) detection kit
Kit of the present invention is liquid double reagent, is divided into reagent 1 and reagent 2, and the volume ratio of reagent 1 and reagent 2 is 3:1.
Specific ingredient is as follows
Reagent R1：Phosphate buffer pH6.0-7.0 0.1-0.5mol/L
PEG 8000 9-15g/L
Sodium azide 0.01wt%
The concrete component preferred embodiment is：
Phosphate buffer, pH6.5 0.2mol/L
PEG 8000 12g/L
Sodium azide 0.01wt%
2 ingredient of the reagent is as follows：
Use embodiment
The use of cardic fatty acid binding protein (h-FABP) detection kit
H-FABP antibody fragment compounded latex particles and cardic fatty acid binding protein (h-FABP) detection kit
It prepares identical as the process being previously mentioned in technical solution.
Detecting instrument：Biochemical Analyzer with 520nm wavelength, 37 DEG C of thermostats.
Test serum：Not haemolysis serum.
Specific detection program：
Calibration procedure：Multiple spot is calibrated, using gamma correction pattern.
As a result it calculates：To measure pipe Δ A, h-FABP contents can be acquired according to calibration curve.
Precision measures：Same sample continuous drawing is measured for 20 times, mean, standard deviation and the change of measured value are calculated
Different coefficient,
1 precision testing result of table
For coefficient of variation CV commonly used in weighing the precision of an assay method, CV values are smaller, indicate 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 to receive
's.CV values are less than 5% in table 1, show that the present invention has preferable precision.
Difference between batch measures：3 batches of kits are prepared respectively, and labeled as 1., 2., 3., each lot number is to same sample test 3
It is secondary, calculate separately the mean value of 3 detections of every batch ofRelative deviation R is calculated as follows.
In formula：
In maximum value；In minimum value；--- 3 batches of reagents detect mean value.
2 difference between batch testing result of table
Difference between batch between 3 lot number kits should be acceptable no more than 10%, and 3 lot numbers of this kit are criticized after testing
Between difference be 5.3%, in the tolerance interval.
Accuracy determination：With same quality-control product, replication 3 times is averaged, should be with quality-control product target value relative deviation
In ± 15% range.
3 accuracy testing result of table
Relative deviation CB=5.33% in table 3 shows that kit of the present invention has excellent standard in ± 15% range
Exactness.
Linear determination：Using deionized water by reagent dilutions at 5 gradient concentrations, each gradient concentration is detected 3 times, is made even
Mean value makees regression analysis to measured value and desired value, calculates r values and relative deviation (the result is shown in Figure 1, unit ng/ml).
4 linear correlation detection result of table
| Diluted concentration | 37.50 | 62.50 | 125.00 | 187.50 | 250.00 |
| Measured value | 36.51 | 59.26 | 128.14 | 186.40 | 242.03 |
| 36.74 | 56.15 | 132.58 | 191.10 | 230.87 | |
| 38.76 | 56.48 | 134.16 | 182.43 | 231.93 | |
| Mean value | 37 | 57 | 132 | 187 | 235 |
| Absolute deviation | 1.81 | 5.65 | 9.20 | 4.72 | 6.46 |
| Relative deviation | 4.63% | 8.98% | - 7.51% | - 2.60% | 2.68% |
Related coefficient is obtained by table 4：r2=0.9935, linear equation is：Y=0.9518x+3.4609, the results showed that
Kit correlation of the present invention is good.
Detect embodiment
Kit of the present invention is compared with the performance indicator of commercial reagent box：
H-FABP antibody fragment compounded latex particles and cardic fatty acid binding protein (h-FABP) detection kit
It prepares identical as the process being previously mentioned in technical solution.
The commercial reagent box of control, information are as follows：
Name of product：(Jinhua is prosperous and powerful for cardic fatty acid binding protein detection kit (latex enhancing immune turbidimetry)
Bio tech ltd)
Dosage form：Liquid double reagent (1：1)
Reagent components：
Reagent 1：
Glycine buffer 100mmol/L
Appropriate bovine serum albumin(BSA)
Appropriate methylisothiazolinone
Reagent 2：
It is coated with the nanoparticle 0.05%-0.5% of cardic fatty acid binding protein antibody
Appropriate bovine serum albumin(BSA)
Appropriate methylisothiazolinone
The method of inspection：
1, reagent prepares：Reagent is to use formula.
2, basic parameter：Method：End-point method sample/R1/R2/：6/100/100
Dominant wavelength：570nm commplementary wave lengths：800nm reaction temperatures：37℃
3, it operates：
4. calculating：△ A=A2-A1
Sample h-FABP (g/L)=△ Au/ △ As x Cs
Commercial reagent box by specification operation.
Linear determination：Kit of the present invention and commercial reagent box is respectively adopted, using AU480 automatic clinical chemistry analyzers,
To 50 parts of samples (including normal and exceptional sample), it is measured by each autoregressive parameter, and correlation analysis (result is carried out to measured value
See Fig. 2, what X-axis indicated is the measured value of kit of the present invention, and what Y-axis indicated is the measured value of commercial reagent box).Phase relation
Number：r2=0.9921, linear equation is：Y=0.976x+0.214, the results showed that kit of the present invention is related to commercial reagent box
Property is good.
5 linear correlation detection result of table
The above disclosure is only the preferred embodiments of the present invention, cannot limit the right model of the present invention with this certainly
It encloses, therefore equivalent changes made in accordance with the claims of the present invention, is still within the scope of the present invention.
Claims (3)
1. a kind of kit of detection cardic fatty acid binding protein, it is characterised in that：Kit includes by reagent 1 and reagent 2
The liquid double reagent component of composition, wherein
1 ingredient of the reagent is as follows：
Phosphate buffer pH6.0-7.0 0.1-0.5mol/L
PEG 8000 9-15g/L
Sodium azide 0.01wt%
2 ingredient of the reagent is as follows：
The volume ratio of the reagent 1 and reagent 2 is 3:1.
2. kit according to claim 1, which is characterized in that the h-FABP antibody fragment compounded latex particles
General following methods prepare：With antibody fragmentation technology, under the buffer environment containing cysteine, first by ficin
Enzyme cuts h-FABP antibody, the peptide fragment of h-FABP antibody F (ab') 2 segment and Fc is formed, through Protein A
Centrifugal column is purified by flash, and removes the peptide fragment of Fc, obtains h-FABP antibody F (ab') 2 segment of purifying, then by h-FABP
2 segments of antibody F (ab') are crosslinking on carboxylated latex microballoon, form h-FABP antibody fragment compounded latex particles.
3. kit according to claim 2, which is characterized in that include the following steps：
(1) h-FABP antibody is cut into 2 segments of F (ab') by ficin；
The anti-human h-FABP antibody of mouse is gone into salt plug desalination with centrifugal, buffer solution used is 0.2mol/L phosphate buffers
(pH6.5)；Identical phosphate buffer 20ml is taken, ficin 1mg, 1mol/L cysteine solution 1ml is added,
0.5mol/L EDTA solution 1ml, are placed in 37 DEG C, uniform dissolution 2h in 100r/min water bath with thermostatic control shaking tables；Cross Sephadex G50
Column is used in combination 0.2mol/L phosphate buffers (pH6.5) to be eluted, and it is molten to collect ficin enzyme of the acquisition containing activation
Liquid, and it is concentrated into original volume；With volume ratio 5：The anti-human h-FABP antibody of mouse after 1 mixing dialysis and activation ficin
Enzyme solutions are placed in 37 DEG C, and 3h is digested in 100r/min water bath with thermostatic control shaking tables, the activation ficin of equivalent is added after 3h again
Enzyme enzyme solutions continue to digest 3h, in triplicate；0.1mmol/L iodoacetamide 0.5ml ice baths 1h is added after enzymolysis to terminate instead
It answers；Protein A centrifugal columns are crossed, the 0.2mol/L phosphate buffers (pH6.5) containing 0.1mol/LNaCl is used in combination to be eluted,
Finally for 24 hours with ultra-pure water dialysis, it collects and obtains h-FABP antibody F (ab') 2 segment, be placed in 2-8 DEG C of preservation；
(2) 2 segments of F (ab') are crosslinking on carboxylated latex microballoon, form h-FABP antibody fragment compounded latex particles；
It is a concentration of 10% latex microsphere 1ml of 120nm to take grain size, and the phosphate buffer 9ml of 0.2mol/L, pH6.5, room is added
Temperature concussion 2mins, is rapidly added 120 μ l EDC aqueous solutions (50mg/ml), 150 μ l N- hydroxy ambers is added after mixing
Amber acid imide aqueous solution (100mg/ml), is stirred at room temperature 40min, and 2-8 DEG C of centrifugation 30min (rotating speed 15000rpm) uses 0.2mol/
L phosphate buffers (pH6.5) wash three times, remove supernatant, precipitation 0.2mol/L phosphate buffers (pH6.5) 1ml
Ultrasound is resuspended；0.5mlh-FABP antibody F (ab') 2 segment is taken, 0.5ml latex microsphere re-suspension liquids are added, 1h is stirred at room temperature, then
It repeats that 0.5ml latex microsphere re-suspension liquids are added, continues that 3h is stirred at room temperature, 400 μ l, 5% hydroxylamine hydrochloride aqueous solutions are added, examination is quenched
Agent and 200 μ l 1%BSA aqueous solutions, room temperature shake 1h；4 DEG C of centrifugation 30min (rotating speed 15000rpm), precipitation 0.2mol/L phosphorus
Phthalate buffer (pH6.5) washs three times, supernatant is removed, with 0.2mol/L phosphate buffers (pH6.5) 1ml ultrasonic disperses
30min, 2-8 DEG C are sealed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810158547.2A CN108490186A (en) | 2018-02-26 | 2018-02-26 | A kind of kit of detection cardic fatty acid binding protein |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810158547.2A CN108490186A (en) | 2018-02-26 | 2018-02-26 | A kind of kit of detection cardic fatty acid binding protein |
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| Publication Number | Publication Date |
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| CN108490186A true CN108490186A (en) | 2018-09-04 |
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ID=63340685
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111487413A (en) * | 2019-01-29 | 2020-08-04 | 艾维可生物科技有限公司 | Detection kit for quantitatively detecting heart-type fatty acid binding protein by E L ISA method |
-
2018
- 2018-02-26 CN CN201810158547.2A patent/CN108490186A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111487413A (en) * | 2019-01-29 | 2020-08-04 | 艾维可生物科技有限公司 | Detection kit for quantitatively detecting heart-type fatty acid binding protein by E L ISA method |
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