CN108037281A - A kind of cardic fatty acid binding protein immunologic function test reagent and its preparation and detection method - Google Patents
A kind of cardic fatty acid binding protein immunologic function test reagent and its preparation and detection method Download PDFInfo
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Abstract
The present invention relates to a kind of cardic fatty acid binding protein immunologic function test reagent and its preparation and detection method, and in particular to a kind of cardioid fat associated proteins immunologic function test reagent and its preparation and detection method, including：Enzyme mark cardioid fat associated proteins, the indicator for detecting cardioid fat associated proteins enzyme-labeled antibody cardioid fat associated proteins compound.The invention has the beneficial effects that：The homogeneous immunological detection reagent of the present invention can easily and fast, accurately determine the protein-bonded content of center of a sample's type fat, and multiple samples can be measured at the same time in automatic clinical chemistry analyzer, realize the protein-bonded rapid measure of high throughput of cardioid fat, accuracy is high, high specificity, stability is good, and accuracy and detection efficiency are enhanced.
Description
Technical field
The present invention relates to a kind of cardic fatty acid binding protein detection reagent and its preparation and detection method, and in particular to one
Kind cardic fatty acid binding protein homogeneous enzyme immunoassay detection reagent and its preparation and detection method.
Background technology
Fatty acid binding protein (fatty acid-binding protein, FABP) is the small molecule egg of one group of multi-source
White matter, is distributed widely in the various kinds of cell kytoplasm such as the mammal heart, brain, bone, intestines, liver, fat, is divided into according to its tissue specificity
Nine types such as cardioid, brain type, kidney type, liver type, visible peristalsis visible intestinal peristalsis.Wherein cardic fatty acid binding protein (Heart-type Fatty
Acid-Binding Protein, H-FABP) cardiac muscle cell is distributed in, the 4%~8% of heart all soluble protein is accounted for,
Energy metabolism of myocardial regulation and control are primarily involved in, because of the difference in its morphology and immunology so that it has height to cardiac muscle cell
Specificity.A kind of soluble protein that H-FABP is made of 132 amino acid, molecular weight are about 15000, it largely exists
It is high to the diagnostic sensitivity of myocardial damage in cardiac muscular tissue, can sensitively it damage reflecting myocardium, being found first within 1988 can
Marker as myocardial damage early stage.Since its molecular weight is small, specificity preferably, quick release when myocardial damage and ischemic,
It is considered as the optimal inspection for the representative-acute myocardial infarction AMI (AMI) for being expected to become early diagnosis acute coronary artery syndrome (ACS)
Survey one of index.1 ~ 3h starts to raise after acute coronary artery syndrome occurs, 6 ~ 8h peakings, and 12 ~ 24h recovers normal, therefore H-
The important symbol thing that FABP can be early diagnosed as ACS, while H-FABP is also a kind of Applications of Cardiac Markers of early diagnosis AMI,
Its specificity and sensitiveness are better than myoglobins (Mb), early diagnosis in AMI, predict the recurrence of AMI, speculate AMI areas,
Whether Reperfu- sion is successful after monitoring AMI, assesses heart failure prognosis etc. is widely used.
At present, Quantitative in vitro measure cardic fatty acid binding protein mainly has electrophoresis and determination of immunological methods.Electrophoresis
Method operation is more numerous and diverse time-consuming, and accuracy and poor repeatability, are not suitable for routine clinical test.Immunological method includes immune electricity
Swimming, radio-immunity, enzyme-linked immunosorbent assay, chemistry or Electrochemiluminescence immunoassay, fluorescent immune method and immune turbidimetry etc..
Immunoelectrophoresis is due to time-consuming longer, unsuitable high throughput detection；Radio-immunity is due to radioactive pollution, quilt substantially
Other methods are substituted；The degree of automation is not high before enzyme-linked immunosorbent assay, so repeatability is not fine, now with
The popularization of the degree of automation, has been greatly improved.Chemistry or Electrochemiluminescence immunoassay, fluorescent immune method are due to needing spy
Different equipment, causes cost higher, use scope is subject to certain restrictions.Scattered light urbidmetry must be on Special Protein Analyzer
Carry out, although precision is preferable, uses and receive certain limitation.
Lack high sensitivity, especially the cardic fatty acid binding protein detection reagent of high specificity, matter currently on the market
Measured Automated inspection reagent.
Homogeneous enzyme immunoassay detection method, with its detection speed is fast, easy to operate, high sensitivity, high specificity and can be complete
The advantages of rapid detection of high throughput to detecting material is realized on automatic biochemistry analyzer, starts more and more to be closed
Note.
The content of the invention
To solve the deficiencies in the prior art, it is an object of the invention to provide a kind of not only safety again can quickly, it is efficient, clever
Cardic fatty acid binding protein homogeneous enzyme immunoassay that is quick, accurately detecting sample to be tested centre type fatty acid binding protein content
Detection reagent and preparation method thereof, and can be combined with various types of automatic biochemistry analyzers, testing staff is required not
High cardic fatty acid binding protein homogeneous enzyme immunoassay detection method.
In order to realize above-mentioned target, the technical solution adopted by the present invention is：
A kind of cardic fatty acid binding protein immunologic function test reagent, it is characterised in that including：Enzyme mark cardioid aliphatic acid combination egg
In vain, for detecting the indicator of cardic fatty acid binding protein antibody-enzyme mark cardic fatty acid binding protein compound.
Above-mentioned indicator is selected from enzymatic reagent, radio isotope reagent, fluorometric reagent or chemical illuminating reagent.
Foregoing cardic fatty acid binding protein immunologic function test reagent, above-mentioned indicator are selected from enzymatic reagent, including：Enzyme mark is even
Join the substrate of thing and enzyme；Above-mentioned enzyme mark conjugate includes glucose dehydrogenase-enzyme-labelled antigen conjugate；The substrate of above-mentioned enzyme is
Glucose.
Foregoing cardic fatty acid binding protein immunologic function test reagent, above-mentioned glucose dehydrogenase-enzyme-labelled antigen conjugate
It is coupled and is formed with cardic fatty acid binding protein by glucose dehydrogenase.
A kind of preparation method of cardic fatty acid binding protein immunologic function test reagent, it is characterised in that include the following steps：
（1）The preparation of glucose dehydrogenase-enzyme-labelled antigen conjugate：Glucose dehydrogenase solution is prepared, activates cardioid aliphatic acid
Associated proteins, make glucose dehydrogenase be coupled with cardic fatty acid binding protein；
（2）The preparation of cardic fatty acid binding protein homogeneous enzyme immunoassay detection reagent：
The preparation of reagent 1：Mixed by cardic fatty acid binding protein antibody and homogeneous zymolyte；
The preparation of reagent 2：Mixed by glucose dehydrogenase-enzyme-labelled antigen conjugate with PBS buffer.
A kind of preparation method of foregoing cardic fatty acid binding protein immunologic function test reagent, the step（1）Specifically
Process is：
1）Glucose dehydrogenase（GDH）The preparation of solution：
A. the GDH that 15mg specifications are 100KU is weighed, room-temperature dissolution contains 72.6mg in 12mL（0.05M）PBS、8mgMgCl2
（3.3mM）In the solution of 100mg NaCl, pH value of solution=8.0；
B. add nicotinamide adenine dinucleotide NADH, the 135mg glucose of 225mg reduction-states and 0.75mL cards must
Alcohol；
C. 2mL dimethyl sulfoxide (DMSO)s are added dropwise.
2）The activation of cardic fatty acid binding protein
A. 10mg cardic fatty acid binding proteins are weighed under anhydrous conditions, are dissolved in 600 μ LDMF；
B. above-mentioned solution temperature drops to 2-8 DEG C；
C. 3 μ L tri-n-butylamines are added；
D. 3 μ L isobutyl chlorocarbonates are added；
E. stir 30 minutes for 2-8 DEG C.
3）The coupling of glucose dehydrogenase and cardic fatty acid binding protein
A. the good cardic fatty acid binding protein solution of above-mentioned activation is slowly added dropwise into glucose dehydrogenase solution；
B. it is stirred overnight for 2-8 DEG C.
A kind of preparation method of foregoing cardic fatty acid binding protein immunologic function test reagent, step（2）Detailed process
It is as follows：
The preparation of reagent 1：By 4.036g（11.25mM）Nicotinamide adenine dinucleotide NAD, 1.711g of oxidation state
（11.25mM）Homogeneous zymolyte is made in the PBS buffer dissolving of glucose 1L 50mM, pH=8.0；By the cardioid fat of preparation
Fat acid binding protein antibody is added in above-mentioned homogeneous zymolyte, and the volume ratio of antibody and homogeneous zymolyte is 1:100~1:10000
；
The preparation of reagent 2：The PBS that the glucose dehydrogenase of preparation-enzyme-labelled antigen conjugate is added to 50mM, pH=8.0 is buffered
In liquid, the volume ratio of above-mentioned conjugate and PBS buffer is 1:100~1:10000.
Utilize the detection method of cardic fatty acid binding protein immunologic function test reagent, it is characterised in that comprise the following steps：
1）Sample to be tested is contacted with cardic fatty acid binding protein antibody；
2）According to the combination situation of sample to be tested centre type fatty acid binding protein and cardic fatty acid binding protein antibody, utilize
The content of indicator judgement sample centre type fatty acid binding protein；The sample to be tested is serum, blood plasma, saliva or urine
Liquid.
The invention has the beneficial effects that：What the present invention was prepared contains above-mentioned enzyme mark cardic fatty acid binding protein
Homogeneous enzyme immunoassay detection examination can easily and fast, accurately determine the content of cardic fatty acid binding protein in sample, and
Multiple samples can be measured at the same time on automatic clinical chemistry analyzer, the high throughput for realizing cardic fatty acid binding protein is quick
Change measure, high sensitivity, high specificity, as a result accurately, raw material are cheap, to the of less demanding of testing staff, are conducive to clinical big
Scale is promoted the use of.
Brief description of the drawings
Fig. 1 is cardic fatty acid binding protein homogeneous enzyme immunoassay reaction normal curve map.
Fig. 2 is cardic fatty acid binding protein homogeneous enzyme immunoassay correlation analysis figure.
Embodiment
The technical solution used in the present invention is：
A kind of cardic fatty acid binding protein homogeneous enzyme immunoassay detection reagent, including：Above-mentioned enzyme mark cardic fatty acid binding protein,
For detecting the indicator of cardic fatty acid binding protein antibody-enzyme mark cardic fatty acid binding protein compound.Instruction examination
Agent is selected from enzymatic reagent, radio isotope reagent, fluorometric reagent or chemical illuminating reagent.Preferably, indicator is enzymatic reagent,
Including：The substrate of enzyme mark conjugate and enzyme.Wherein, enzyme mark conjugate includes glucose dehydrogenase-enzyme-labelled antigen conjugate, its
It can be obtained by chemical synthesis process.
The application method of above-mentioned cardic fatty acid binding protein homogeneous enzyme immunoassay detection reagent, comprises the following steps：
1）Sample to be tested is contacted with cardic fatty acid binding protein antibody；
2）According to the combination situation of sample to be tested centre type fatty acid binding protein and above-mentioned cardic fatty acid binding protein antibody,
Utilize the content of indicator judgement sample centre type fatty acid binding protein.
Sample to be tested is various physiology samples, such as serum, blood plasma, urine, saliva etc..Preferably, sample to be tested is blood
Clear or blood plasma.
With reference to specific embodiment, the present invention is further illustrated.
Embodiment one：The preparation of glucose dehydrogenase-enzyme-labelled antigen conjugate
1）Glucose dehydrogenase（GDH）The preparation of solution：
A. the GDH that 15mg specifications are 100KU is weighed, room-temperature dissolution contains 72.6mg in 12mL（0.05M）PBS、8mg MgCl2
（3.3mM）In the solution of 100mg NaCl, pH value of solution=8.0；This step carries out in small beaker A.
In above-mentioned beaker A add 225mg reduction-states nicotinamide adenine dinucleotide NADH, 135mg glucose with
And 0.75mL carbitols.
2mL dimethyl sulfoxide (DMSO)s (dimethy sulfoxide, DMSO) are added dropwise again in above-mentioned beaker A.
2）The activation of cardic fatty acid binding protein
A. 10mg cardic fatty acid binding proteins are weighed under anhydrous conditions, are dissolved in 600 μ LDMF；
B. above-mentioned solution temperature drops to 2-8 DEG C；
C. 3 μ L tri-n-butylamines are added；
D. 3 μ L isobutyl chlorocarbonates are added；
E. stir 30 minutes for 2-8 DEG C；
3）The coupling of glucose dehydrogenase and cardic fatty acid binding protein
A. the good cardic fatty acid binding protein solution of above-mentioned activation is slowly added dropwise into glucose dehydrogenase solution；
B. it is stirred overnight for 2-8 DEG C.
Embodiment two：The preparation of cardic fatty acid binding protein homogeneous enzyme immunoassay detection reagent
Cardic fatty acid binding protein homogeneous enzyme immunoassay detection reagent, including：Above-mentioned enzyme mark cardic fatty acid binding protein, be used for
Detect the indicator of cardic fatty acid binding protein antibody-enzyme mark cardic fatty acid binding protein compound.Indicator selects
From enzymatic reagent, radio isotope reagent, fluorometric reagent or chemical illuminating reagent.Preferably, indicator is enzymatic reagent, bag
Include：The substrate of enzyme mark conjugate and enzyme.Wherein, enzyme mark conjugate includes glucose dehydrogenase-enzyme-labelled antigen conjugate, it is logical
Above-mentioned chemical synthesis process is crossed to obtain.
Cardic fatty acid binding protein homogeneous enzyme immunoassay detection reagent before the use, in order to avoid the enzyme in indicator
Mark conjugate and the substrate of enzyme react, and the substrate of enzyme mark conjugate and enzyme is separated, is not mixed, so by enzyme
Substrate is mixed with above-mentioned cardic fatty acid binding protein antibody.That is, the homogeneous enzyme of cardic fatty acid binding protein
Immunologic function test reagent includes two kinds of reagents being provided separately, specific as follows：
The preparation of reagent 1：By 4.036g（11.25mM）Nicotinamide adenine dinucleotide NAD, 1.711g of oxidation state
（11.25mM）Homogeneous zymolyte is made in the PBS buffer dissolving of glucose 1L 50mM, pH=8.0；By the cardioid fat of preparation
Fat acid binding protein antibody is added in above-mentioned homogeneous zymolyte, and the volume ratio of antibody and homogeneous zymolyte is 1:100~1:10000
, specific ratio is 1 in the present embodiment:500.
The preparation of reagent 2：The glucose dehydrogenase of preparation-enzyme-labelled antigen conjugate is added to the PBS of 50mM, pH=8.0
In buffer solution, the volume ratio of above-mentioned conjugate and PBS buffer is 1:100~1:10000, specific ratio in the present embodiment
For 1:500.
The application method of above-mentioned cardic fatty acid binding protein homogeneous enzyme immunoassay detection reagent, comprises the following steps：
1）Sample to be tested is contacted with cardic fatty acid binding protein antibody；
2）According to the combination situation of sample to be tested centre type fatty acid binding protein and above-mentioned cardic fatty acid binding protein antibody,
Utilize the content of indicator judgement sample centre type fatty acid binding protein.
Specifically, during detection, sample to be tested is added in reagent 1, the cardic fatty acid binding protein in sample to be tested
Specifically bound with the cardic fatty acid binding protein antibody in reagent 1, generation cardic fatty acid binding protein antibody-
Cardic fatty acid binding protein compound；Reagent 2 is added, at this time the glucose dehydrogenase in reagent 2-enzyme-labelled antigen coupling
Thing is mixed with the substrate of the enzyme in reagent 1, contacted, generation enzymatic reaction, and composition detection cardic fatty acid binding protein antibody-
The indicator of cardic fatty acid binding protein compound, indicator according to sample to be tested centre type fatty acid binding protein with
The combination situation of above-mentioned cardic fatty acid binding protein antibody judges the content of sample to be tested centre type fatty acid binding protein.
Since the cardic fatty acid binding protein in glucose dehydrogenase-enzyme-labelled antigen conjugate and sample to be tested competes
Property combination cardic fatty acid binding protein antibody, so, the content of sample to be tested centre type fatty acid binding protein is more, homogeneously
The amount of the glucose dehydrogenase-enzyme-labelled antigen conjugate to dissociate in enzyme solutions is more, and enzymatic reaction is faster, causes on OD340
Rise.
Above-mentioned sample to be tested is physiology sample, such as serum, blood plasma, urine, saliva etc..
As a preferred solution, above-mentioned sample to be tested is serum or blood plasma.
Embodiment three：Cardic fatty acid binding protein homogeneous enzyme immunoassay is examined
1st, standard curve is obtained：Olympus AU400 automatic clinical chemistry analyzer response parameters are set, and operating process is：First plus
Reagent 1, adds standard items, is eventually adding reagent 2.After adding reagent 2, the OD340 light absorption values of different time points are measured, are calculated
Go out reaction rate during various criterion product concentration, the constantly volume ratio of adjustment reagent 1 and reagent 2 needed in actual mechanical process,
Survey luminous point is adjusted at the same time, comparatively ideal reaction normal curve map is finally drawn, as shown in Fig. 1.
2nd, Biochemical Analyzer detects：By taking the operation of Olympus AU400 automatic clinical chemistry analyzers as an example：8 μ L samples are added,
Then 160 μ L reagents 1 are added, blending incubation 5min, adds 40 μ L reagents 2, blending incubation 30s, then starts to read absorbance
A1, then after being incubated 5min, absorbance A 2 is read, calculate Δ A=A2-A1.Reaction condition：Dominant wavelength：700nm；Reaction temperature：37
℃；Methodology：End-point method；The Direction of Reaction：Rise；Calibration mode：Multiple spot is non-linear.Calibration object concentration：0.00,15.00,
30.00,60.00,120.00 ng/mL.
The standard curve obtained by the homogeneous enzyme immunoassay detection reagent of the present invention, replication is low, high concentration Quality Control sample
Sheet 10 times, above-mentioned Quality Control sample are：Cardic fatty acid binding protein standard items are dissolved in human serum, are respectively to concentration
30.00 120.00 ng/mL.Detection data and data analysis are shown in Table 1.
1 sample precision of table is assessed
| Blood sample | It is low | It is high |
| Concentration of specimens（ng/mL） | 30.00 | 120.00 |
| 1 | 28.88 | 120.49 |
| 2 | 30.86 | 118.69 |
| 3 | 30.16 | 123.62 |
| 4 | 30.91 | 116.18 |
| 5 | 28.68 | 120.66 |
| 6 | 28.04 | 121.77 |
| 7 | 29.78 | 118.15 |
| 8 | 30.97 | 120.58 |
| 9 | 29.92 | 121.03 |
| 10 | 28.78 | 117.36 |
| Average value（ng/mL） | 29.70 | 119.85 |
| Standard deviation（SD） | 1.05 | 2.23 |
| Precision（CV%） | 3.55 | 1.86 |
Testing result：The accuracy of the homogeneous enzyme immunoassay detection reagent measure of the present invention is high, and precision is high, and CV is below 4%.
Example IV：Correlation analysis
60 clinical samples including 40 positive samples and 20 ' negative ' specimens are used with the biological skill of member in Chongqing respectively
The immunochromatographic method of art Co., Ltd and the homogeneous enzyme immunoassay method of the present invention carry out correlation analysis, and the data of measure are referring to table 2.
2 authentic specimen measured value of table
| Catalogue number(Cat.No.) | Homogeneous enzyme immunoassay method measured value（ng/mL） | Immunochromatographic method measured value（ng/mL） |
| 1 | 3.98 | 3.54 |
| 2 | 3.48 | 2.07 |
| 3 | 113.56 | 110.00 |
| 4 | 4.73 | 4.35 |
| 5 | 3.51 | 3.73 |
| 6 | 3.47 | 2.98 |
| 7 | 3.83 | 3.00 |
| 8 | 3.41 | 2.70 |
| 9 | 3.02 | 2.77 |
| 10 | 3.27 | 3.65 |
| 11 | 3.66 | 3.69 |
| 12 | 3.94 | 2.07 |
| 13 | 3.57 | 3.83 |
| 14 | 3.85 | 2.52 |
| 15 | 3.63 | 2.38 |
| 16 | 63.20 | 64.06 |
| 17 | 3.83 | 3.17 |
| 18 | 3.78 | 2.03 |
| 19 | 3.24 | 3.57 |
| 20 | 3.76 | 3.13 |
| 21 | 3.52 | 2.97 |
| 22 | 3.70 | 2.36 |
| 23 | 3.02 | 2.21 |
| 24 | 23.04 | 22.42 |
| 25 | 3.95 | 2.43 |
| 26 | 81.42 | 83.57 |
| 27 | 3.36 | 2.41 |
| 28 | 50.74 | 52.38 |
| 29 | 3.91 | 3.43 |
| 30 | 3.48 | 3.22 |
| 31 | 36.58 | 38.28 |
| 32 | 3.39 | 2.23 |
| 33 | 23.33 | 25.04 |
| 34 | 3.77 | 3.64 |
| 35 | 3.97 | 3.83 |
| 36 | 72.13 | 70.24 |
| 37 | 3.38 | 3.02 |
| 38 | 90.23 | 92.26 |
| 39 | 3.79 | 3.68 |
| 40 | 63.01 | 61.73 |
| 41 | 3.69 | 3.56 |
| 42 | 45.29 | 43.58 |
| 43 | 3.44 | 2.17 |
| 44 | 8.02 | 9.27 |
| 45 | 3.93 | 3.01 |
| 46 | 41.79 | 43.51 |
| 47 | 3.29 | 3.80 |
| 48 | 73.65 | 72.03 |
| 49 | 18.38 | 17.80 |
| 50 | 3.13 | 2.74 |
| 51 | 95.98 | 92.44 |
| 52 | 3.67 | 3.02 |
| 53 | 20.47 | 22.48 |
| 54 | 3.25 | 2.37 |
| 55 | 82.07 | 84.35 |
| 56 | 3.94 | 3.09 |
| 57 | 30.15 | 31.85 |
| 58 | 3.24 | 3.12 |
| 59 | 2.45 | 2.56 |
| 60 | 53.88 | 52.72 |
Map to above-mentioned data, referring to Fig. 2, obtained linear equation is：Y=0.9812x+0.5830, coefficient R2=
0.9960, show that the Troponin I clinical samples accuracy of the detection reagent measure of the present invention is high.
Since the detection process of the present invention is completed by instrument is full-automatic, so to the of less demanding of testing staff, easily
In realizing and promote the use of.
It should be noted that the foregoing is merely the embodiment of the present invention, it is not intended to limit the scope of the invention,
Every equivalent structure or equivalent flow shift done using description of the invention and accompanying drawing content, is directly or indirectly used in
Other correlative technology fields, are included within the scope of the present invention.
Claims (7)
- A kind of 1. cardic fatty acid binding protein homogeneous enzyme immunoassay detection reagent, it is characterised in that including：Enzyme mark cardioid aliphatic acid Associated proteins, the instruction for anti-detection cardic fatty acid binding protein antibody-enzyme mark cardic fatty acid binding protein compound Reagent.
- 2. the cardic fatty acid binding protein detection reagent according to claim 1, it is characterised in that：Above-mentioned enzyme mark coupling Thing is coupled with cardic fatty acid binding protein by glucose dehydrogenase and is formed including glucose dehydrogenase-enzyme-labelled antigen conjugate.
- 3. the cardic fatty acid binding protein detection reagent according to claim 1, it is characterised in that：Above-mentioned indicator choosing From enzymatic reagent, the enzymatic reagent is made of the substrate of enzyme mark conjugate and enzyme, and enzyme mark conjugate is glucose dehydrogenase-enzyme mark Antigen conjugates, the substrate of enzyme is glucose.
- 4. a kind of cardic fatty acid binding protein immunologic function test reagent, it is characterised in that include the following steps：（1）The preparation of glucose dehydrogenase-enzyme-labelled antigen conjugate：Glucose dehydrogenase solution is prepared, activates cardioid aliphatic acid Associated proteins, make glucose dehydrogenase be coupled with cardic fatty acid binding protein；（2）The preparation of cardic fatty acid binding protein homogeneous immunological detection reagent：The preparation of reagent 1：Mixed by cardic fatty acid binding protein antibody and homogeneous zymolyte；The preparation of reagent 2：Mixed by glucose dehydrogenase-enzyme-labelled antigen conjugate with phosphate buffer.
- 5. a kind of preparation method of cardic fatty acid binding protein immunologic function test reagent according to claim 4, its feature It is, the step（1）Detailed process is：Glucose dehydrogenase（GDH）The preparation of solution：A. the GDH that 15mg specifications are 100KU is weighed, room-temperature dissolution contains 72.6mg in 12mL（0.05M）PBS、8mgMgCl2 （3.3mM）In the solution of 100mg NaCl, pH value of solution=8.0；B. add nicotinamide adenine dinucleotide NADH, the 135mg glucose of 225mg reduction-states and 0.75mL cards must Alcohol；C. 2mL dimethyl sulfoxide (DMSO)s are added dropwise2）The activation of cardic fatty acid binding proteinA. 10mg cardic fatty acid binding proteins are weighed under anhydrous conditions, are dissolved in 600 μ LDMF；B. above-mentioned solution temperature drops to 2-8 DEG C；C. 3 μ L tri-n-butylamines are added；D. 3 μ L isobutyl chlorocarbonates are added；E. stir 30 minutes for 2-8 DEG C3）The coupling of glucose dehydrogenase and cardic fatty acid binding proteinA. the good cardic fatty acid binding protein solution of above-mentioned activation is slowly added dropwise into glucose dehydrogenase solution；B. it is stirred overnight for 2-8 DEG C.
- 6. a kind of preparation method of cardic fatty acid binding protein homogeneous enzyme immunoassay detection reagent according to claim 4, It is characterized in that, step（2）Detailed process it is as follows：The preparation of reagent 1：By 4.036g（11.25mM）Nicotinamide adenine dinucleotide NAD, 1.711g of oxidation state （11.25mM）Homogeneous zymolyte is made in the PBS buffer dissolving of glucose 1L 50mM, pH=8.0；By the cardioid fat of preparation Fat acid binding protein antibody is added in above-mentioned homogeneous zymolyte, and the volume ratio of antibody and homogeneous zymolyte is 1:100~1:10000 ；The preparation of reagent 2：The PBS that the glucose dehydrogenase of preparation-enzyme-labelled antigen conjugate is added to 50mM, pH=8.0 is buffered In liquid, the volume ratio of above-mentioned conjugate and PBS buffer is 1:100~1:10000.
- 7. the detection method of the cardic fatty acid binding protein immunologic function test reagent described in 3 any one of claims 1 to 3 is utilized, It is characterised in that it includes following steps：1）Sample to be tested is contacted with cardic fatty acid binding protein antibody；2）According to the combination situation of sample to be tested centre type fatty acid binding protein and cardic fatty acid binding protein antibody,Utilize the content of indicator judgement sample centre type fatty acid binding protein；The sample to be tested is serum, blood plasma, saliva Liquid or urine.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111363035A (en) * | 2018-12-25 | 2020-07-03 | 东莞市朋志生物科技有限公司 | Recombinant antibody of anti-heart fatty acid binding protein |
| CN111363036A (en) * | 2018-12-25 | 2020-07-03 | 东莞市朋志生物科技有限公司 | Recombinant antibody of anti-heart fatty acid binding protein |
| CN115267171A (en) * | 2022-06-24 | 2022-11-01 | 北京丹大生物技术有限公司 | Reagent, kit and method for detecting aripiprazole and dehydroaripiprazole concentrations based on homogeneous enzymatic method |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111363035A (en) * | 2018-12-25 | 2020-07-03 | 东莞市朋志生物科技有限公司 | Recombinant antibody of anti-heart fatty acid binding protein |
| CN111363036A (en) * | 2018-12-25 | 2020-07-03 | 东莞市朋志生物科技有限公司 | Recombinant antibody of anti-heart fatty acid binding protein |
| CN111363036B (en) * | 2018-12-25 | 2021-10-12 | 东莞市朋志生物科技有限公司 | Recombinant antibody of anti-heart fatty acid binding protein |
| CN111363035B (en) * | 2018-12-25 | 2021-12-03 | 东莞市朋志生物科技有限公司 | Recombinant antibody of anti-heart fatty acid binding protein |
| CN115267171A (en) * | 2022-06-24 | 2022-11-01 | 北京丹大生物技术有限公司 | Reagent, kit and method for detecting aripiprazole and dehydroaripiprazole concentrations based on homogeneous enzymatic method |
| CN115267171B (en) * | 2022-06-24 | 2025-01-07 | 北京丹大生物技术有限公司 | A reagent, a kit and a method for detecting the concentration of aripiprazole and dehydro-aripiprazole based on a homogeneous enzyme method |
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